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Sample records for 10-deacetylbaccatin iii-10-o-acetyl transferase

  1. Roles for glutathione transferases in antioxidant recycling

    PubMed Central

    Dixon, David P; Steel, Patrick G

    2011-01-01

    Uniquely among the plant glutathione transferases, two classes possess a catalytic cysteine capable of performing glutathione-dependent reductions. These are the dehydroascorbate reductases (DHARs) and the lambda-class glutathione transferases (GSTLs). Using immobilized GSTLs probed with crude plant extracts we have identified flavonols as high affinity ligands and subsequently demonstrated a novel glutathione-dependent role for these enzymes in recycling oxidized quercetin. By comparing the activities of DHARs and GSTLs we now propose a unified catalytic mechanism that suggests oxidized anthocyanidins and tocopherols may be alternative polyphenolic substrates of GSTLs. PMID:21778824

  2. Purification and characterization of the Oligosaccharyl transferase

    SciTech Connect

    Kapoor, T.M.

    1990-11-01

    Oligosaccharyl transferase was characterized to be a glycoprotein with at least one saccharide unit that had a D-manno or D- glucopyranose configuration with unmodified hydroxy groups at C-3, C-4 and C-6, using a Concanavalin A affinity column. This afforded a 100 fold increase in the transferase purity in the solubilized microsomal sample and also removed over 90% of the microsomal proteins (the cytosolic ones being removed before solubilization). The detergent, N,N-Dimethyldodecylamine N-oxide (LDAO) was used for solubilization and it yielded a system compatible with the assay and the purification steps. An efficient method for detergent extraction without dilution of sample or protein precipitation was also developed.

  3. Nomenclature for mammalian soluble glutathione transferases.

    PubMed

    Mannervik, Bengt; Board, Philip G; Hayes, John D; Listowsky, Irving; Pearson, William R

    2005-01-01

    The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs. PMID:16399376

  4. Single prenyl-binding site on protein prenyl transferases

    PubMed Central

    Desnoyers, Luc; Seabra, Miguel C.

    1998-01-01

    Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions. PMID:9770475

  5. The Genetic Architecture of Murine Glutathione Transferases

    PubMed Central

    Lu, Lu; Pandey, Ashutosh K.; Houseal, M. Trevor; Mulligan, Megan K.

    2016-01-01

    Glutathione S-transferase (GST) genes play a protective role against oxidative stress and may influence disease risk and drug pharmacokinetics. In this study, massive multiscalar trait profiling across a large population of mice derived from a cross between C57BL/6J (B6) and DBA2/J (D2)—the BXD family—was combined with linkage and bioinformatic analyses to characterize mechanisms controlling GST expression and to identify downstream consequences of this variation. Similar to humans, mice show a wide range in expression of GST family members. Variation in the expression of Gsta4, Gstt2, Gstz1, Gsto1, and Mgst3 is modulated by local expression QTLs (eQTLs) in several tissues. Higher expression of Gsto1 in brain and liver of BXD strains is strongly associated (P < 0.01) with inheritance of the B6 parental allele whereas higher expression of Gsta4 and Mgst3 in brain and liver, and Gstt2 and Gstz1 in brain is strongly associated with inheritance of the D2 parental allele. Allele-specific assays confirmed that expression of Gsto1, Gsta4, and Mgst3 are modulated by sequence variants within or near each gene locus. We exploited this endogenous variation to identify coexpression networks and downstream targets in mouse and human. Through a combined systems genetics approach, we provide new insight into the biological role of naturally occurring variants in GST genes. PMID:26829228

  6. Detection of glutathione transferase activity on polyacrylamide gels.

    PubMed

    Ricci, G; Lo Bello, M; Caccuri, A M; Galiazzo, F; Federici, G

    1984-12-01

    A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues. PMID:6532239

  7. Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver.

    PubMed Central

    Awasthi, Y C; Dao, D D; Saneto, R P

    1980-01-01

    Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity. Images Fig. 3. PMID:7470087

  8. Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase

    PubMed Central

    Motea, Edward A.; Berdis, Anthony J.

    2009-01-01

    Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis. PMID:19596089

  9. Inhibition of hepatic glutathione transferases by propylthiouracil and its metabolites.

    PubMed

    Kariya, K; Sawahata, T; Okuno, S; Lee, E

    1986-05-01

    The effects of propylthiouracil (PTU) and its metabolites on the activity of GSH transferases were examined using rat liver cytosol. PTU inhibited the enzyme activity toward both CDNB and DCNB in a concentration-dependent manner. At the concentration of 10 mM, PTU caused 25% inhibition, which was the maximum effect. PTU derivatives such as propyluracil and thiouracil showed the same effect as the parent compound. On the other hand, S-oxides of PTU such as PTU-SO2 and PTU-SO3, which were chemically synthesized by the oxidation of PTU, were more potent inhibitors of GSH transferases than the parent PTU. A significant inhibition was observed at a concentration of 0.1 mM of PTU S-oxides. At a concentration of 10 mM the S-oxides caused an 80% inhibition of the enzyme activity. PTU inhibited the transferase activity by competing with GSH but the S-oxides of PTU acted by another mechanism. In contrast to the effect on GSH transferases, PTU-SO3 had a weak inhibitory effect on GSH peroxidase activity. Thus, oxidation of PTU leads to products which are potent inhibitors of GSH transferases. PMID:3707612

  10. Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors

    PubMed Central

    Kosa, Nicolas M.; Foley, Timothy L.; Burkart, Michael D.

    2016-01-01

    Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity. PMID:24192555

  11. Thioltransferase activity of bovine lens glutathione S-transferase.

    PubMed Central

    Dal Monte, M; Cecconi, I; Buono, F; Vilardo, P G; Del Corso, A; Mura, U

    1998-01-01

    A Mu-class glutathione S-transferase purified to electrophoretic homogeneity from bovine lens displayed thioltransferase activity, catalysing the transthiolation reaction between GSH and hydroxyethyldisulphide. The thiol-transfer reaction is composed of two steps, the formation of GSSG occurring through the generation of an intermediate mixed disulphide between GSH and the target disulphide. Unlike glutaredoxin, which is only able to catalyse the second step of the transthiolation process, glutathioneS-transferase catalyses both steps of the reaction. Data are presented showing that bovine lens glutathione S-transferase and rat liver glutaredoxin, which was used as a thioltransferase enzyme model, can operate in synergy to catalyse the GSH-dependent reduction of hydroxyethyldisulphide. PMID:9693102

  12. METAL-INDUCED INHIBITION OF GLUTATHIONE S-TRANSFERASES

    EPA Science Inventory

    The glutathione S-transferases comprise a group of multi-functional enzymes involved in the biotransformation/detoxication of a broad spectrum of hydrophobic compounds bearing an electrophilic center. The enzymes facilitate the nucleophilic attack of the -SH group of reduced glut...

  13. Rational design of an organometallic glutathione transferase inhibitor

    SciTech Connect

    Ang, W.H.; Parker, L.J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C.J.; LoBello, M.; Parker, M.W.; Dyson, P.J.

    2010-08-17

    A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor. The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

  14. GLUTATHIONE S-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE

    EPA Science Inventory

    GLUTATHIONE s-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE. M K Ross1 and R A Pegram2. 1Curriculum in Toxicology, University of North Carolina at Chapel Hill; 2Experimental Toxicology Division, NHEERL/ORD, United States Environmental Protection Agency, Research Triangl...

  15. 21 CFR 862.1535 - Ornithine carbamyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1535 Ornithine...

  16. Genetics Home Reference: succinyl-CoA:3-ketoacid CoA transferase deficiency

    MedlinePlus

    ... CoA:3-ketoacid CoA transferase deficiency succinyl-CoA:3-ketoacid CoA transferase deficiency Enable Javascript to view ... PDF Open All Close All Description Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inherited ...

  17. Developmental aspects of glutathione S-transferase B (ligandin) in rat liver.

    PubMed Central

    Hales, B F; Neims, A H

    1976-01-01

    The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood. PMID:1008852

  18. Purification and characterization of a DNA strand transferase from broccoli.

    PubMed

    Tissier, A F; Lopez, M F; Signer, E R

    1995-05-01

    A protein with DNA binding, renaturation, and strand-transfer activities has been purified to homogeneity from broccoli (Brassica oleracea var italica). The enzyme, broccoli DNA strand transferase, has a native molecular mass of at least 200 kD and an apparent subunit molecular mass of 95 kD and is isolated as a set of isoforms differing only in charge. All three activities are saturated at very low stoichiometry, one monomer per approximately 1000 nucleotides of single-stranded DNA. Strand transfer is not effected by nuclease activity and reannealing, is only slightly dependent on ATP, and is independent of added Mg2+. Transfer requires homologous single- and double-stranded DNA and at higher enzyme concentrations results in very high molecular mass complexes. As with Escherichia coli RecA, transfer by broccoli DNA strand transferase depends strongly on the presence of 3' homologous ends. PMID:7784508

  19. Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

    PubMed

    Auler, Kasie; Broock, Robyn; Nyhan, William L

    2015-01-01

    Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. PMID:26132002

  20. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  1. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  2. Glutathione transferase mimics: micellar catalysis of an enzymic reaction.

    PubMed Central

    Lindkvist, B; Weinander, R; Engman, L; Koetse, M; Engberts, J B; Morgenstern, R

    1997-01-01

    Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as alpha,beta-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium - co - dodecylmethyldiallylammonium) bromide (86/14) >>cetyltrimethylammonium bromide>zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate)>zwittergent+ ++ 3-14 (n-tetradecyl-N,N-dimethyl - 3 - ammonio -1 - propanesulphonate) approximately N,N - dimethyl - laurylamine N-oxide>N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to alpha,beta-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding. PMID:9173899

  3. Nucleotidyl transferase assisted DNA labeling with different click chemistries

    PubMed Central

    Winz, Marie-Luise; Linder, Eva Christina; André, Timon; Becker, Juliane; Jäschke, Andres

    2015-01-01

    Here, we present a simple, modular and efficient strategy that allows the 3′-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3′-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation. PMID:26013812

  4. [Selective N-heterylazimine inhibition of reactions catalyzed by rat liver glutathione transferase].

    PubMed

    Stulovskiĭ, A V; Voznyĭ, I V; Rozengart, E V; Suvorov, A A; Khovanskikh, A E

    1992-01-01

    Three reactions (nucleophile substitution, thiolysis and N-deoxygenation) catalyzed by rat liver glutathione transferase have been studied using several N-heterylazimine inhibitors. The inhibitors are sharply different in their effectiveness in the transferase reactions. Their efficiency depends on their structure. The mechanism which underlies the found regularities is suggested. PMID:1413125

  5. Glutathione S-transferase class {pi} polymorphism in baboons

    SciTech Connect

    Aivaliotis, M.J.; Cantu, T.; Gilligan, R.

    1995-02-01

    Glutathione S-transferase (GST) comprises a family of isozymes with broad substrate specificities. One or more GST isozymes are present in most animal tissues and function in several detoxification pathways through the conjugation of reduced glutathione with various electrophiles, thereby reducing their potential toxicity. Four soluble GST isozymes encoded by genes on different chromosomes have been identified in humans. The acidic class pi GST, GSTP (previously designated GST-3), is widely distributed in adult tissues and appears to be the only GST isozyme present in leukocytes and placenta. Previously reported electrophoretic analyses of erythrocyte and leukocyte extracts revealed single bands of activity, which differed slightly in mobility between the two cell types, or under other conditions, a two-banded pattern. To our knowledge, no genetically determined polymorphisms have previously been reported in GSTP from any species. We now report a polymorphism of GSTP in baboon leukocytes, and present family data that verifies autosomal codominant inheritance. 14 refs., 2 figs., 1 tab.

  6. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  7. Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

    PubMed

    Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

    2005-01-01

    The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase. PMID:15619514

  8. Crystal structure of E. coli lipoprotein diacylglyceryl transferase.

    PubMed

    Mao, Guotao; Zhao, Yan; Kang, Xusheng; Li, Zhijie; Zhang, Yan; Wang, Xianping; Sun, Fei; Sankaran, Krishnan; Zhang, Xuejun C

    2016-01-01

    Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure-function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer. PMID:26729647

  9. Inactivation of Anopheles gambiae Glutathione Transferase ε2 by Epiphyllocoumarin

    PubMed Central

    Marimo, Patience; Hayeshi, Rose; Mukanganyama, Stanley

    2016-01-01

    Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 μM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters kinact and KI were found to be 0.020 ± 0.001 min−1 and 7.5 ± 2.1 μM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs. PMID:26925266

  10. Crystal structure of E. coli lipoprotein diacylglyceryl transferase

    PubMed Central

    Mao, Guotao; Zhao, Yan; Kang, Xusheng; Li, Zhijie; Zhang, Yan; Wang, Xianping; Sun, Fei; Sankaran, Krishnan; Zhang, Xuejun C.

    2016-01-01

    Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure–function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer. PMID:26729647

  11. Benzene oxide is a substrate for glutathione S-transferases.

    PubMed

    Zarth, Adam T; Murphy, Sharon E; Hecht, Stephen S

    2015-12-01

    Benzene is a known human carcinogen which must be activated to benzene oxide (BO) to exert its carcinogenic potential. BO can be detoxified in vivo by reaction with glutathione and excretion in the urine as S-phenylmercapturic acid. This process may be catalyzed by glutathione S-transferases (GSTs), but kinetic data for this reaction have not been published. Therefore, we incubated GSTA1, GSTT1, GSTM1, and GSTP1 with glutathione and BO and quantified the formation of S-phenylglutathione. Kinetic parameters were determined for GSTT1 and GSTP1. At 37 °C, the putative Km and Vmax values for GSTT1 were 420 μM and 450 fmol/s, respectively, while those for GSTP1 were 3600 μM and 3100 fmol/s. GSTA1 and GSTM1 did not exhibit sufficient activity for determination of kinetic parameters. We conclude that GSTT1 is a critical enzyme in the detoxification of BO and that GSTP1 may also play an important role, while GSTA1 and GSTM1 seem to be less important. PMID:26554337

  12. Modulation of Rab GTPase function by a protein phosphocholine transferase.

    PubMed

    Mukherjee, Shaeri; Liu, Xiaoyun; Arasaki, Kohei; McDonough, Justin; Galán, Jorge E; Roy, Craig R

    2011-09-01

    The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions. PMID:21822290

  13. Glutathione S-transferase activity and glutathione S-transferase mu expression in subjects with risk for colorectal cancer.

    PubMed

    Szarka, C E; Pfeiffer, G R; Hum, S T; Everley, L C; Balshem, A M; Moore, D F; Litwin, S; Goosenberg, E B; Frucht, H; Engstrom, P F

    1995-07-01

    The glutathione S-transferases (alpha, mu, and pi), a family of Phase II detoxication enzymes, play a critical role in protecting the colon mucosa by catalyzing the conjugation of dietary carcinogens with glutathione. We investigated the efficacy of using the glutathione S-transferase (GST) activity of blood lymphocytes and GST-mu expression as biomarkers of risk for colorectal cancer. GST activity was measured in the blood lymphocytes of control individuals (n = 67) and in the blood lymphocytes (n = 60) and colon tissue (n = 34) of individuals at increased risk for colon cancer. Total GST activity was determined spectrophotometrically with the use of 1-chloro-2,4-dinitrobenzene as a substrate. The ability to express the um subclass of GST was determined with the use of an ELISA. Although interindividual variability in the GST activity of blood lymphocytes was greater than 8-fold (range, 16.7-146.8 nmol/min/mg), the GST activity of blood lymphocytes and colon tissue within an individual was constant over time and was unrelated to sex, age, or race. The GST activity of blood lymphocytes from high-risk individuals was significantly lower than that of blood lymphocytes from control individuals (P < or = 0.004). No association was observed between the frequency of GST-mu phenotype and risk for colorectal cancer. Blood lymphocytes from high-risk individuals unable to express GST-mu had lower levels of GST activity than did those from control subjects with the GST-mu null phenotype; however, this difference was significant in male subjects only (P < or = 0.006). Analysis of paired samples of blood lymphocytes and colon tissue indicated a strong correlation between the GST activity of the two tissue types (Spearman's rank correlation, r = 0.87; P < or = 0.0001). The GST activity of blood lymphocytes may be used to identify high-risk individuals with decreased protection from this Phase II detoxication enzyme who may benefit from clinical trials evaluating GST modulators

  14. Analysis of Arabidopsis glutathione-transferases in yeast.

    PubMed

    Krajewski, Matthias P; Kanawati, Basem; Fekete, Agnes; Kowalski, Natalie; Schmitt-Kopplin, Philippe; Grill, Erwin

    2013-07-01

    The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and

  15. Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

    PubMed Central

    't Hoen, Peter A C; Out, Ruud; Commandeur, Jan N M; Vermeulen, Nico P E; van Batenburg, F H D; Manoharan, Muthiah; van Berkel, Theo J C; Biessen, Erik A L; Bijsterbosch, Martin K

    2002-01-01

    The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression. PMID:12515389

  16. Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase*

    PubMed Central

    Baron, Rudi A.; Tavaré, Richard; Figueiredo, Ana C.; Błażewska, Katarzyna M.; Kashemirov, Boris A.; McKenna, Charles E.; Ebetino, Frank H.; Taylor, Adam; Rogers, Michael J.; Coxon, Fraser P.; Seabra, Miguel C.

    2009-01-01

    Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases. PMID:19074143

  17. Post-transcriptional regulation of chloramphenicol acetyl transferase.

    PubMed Central

    Byeon, W H; Weisblum, B

    1984-01-01

    The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) mRNA encoded by plasmid pC194 was determined experimentally by using [alpha-32P]ATP-labeled runoff transcripts partially digested with T1 RNase. By partial digestion of the in vitro transcripts with S1, T1, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions, which contain the complementary sequences CCUCC and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12-base-pair inverted complementary repeat sequence (-19.5 kcal [ca. -81.7 kJ] per mol). The CAT mRNA was stable to digestion by T1 RNase at the four guanosine residues in the Shine and Dalgarno sequence GGAGG , even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194 , induced with chloramphenicol, contain about the same concentration of pulse-labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the non- acetylatable chloramphenicol analog fluorothiamphenicol was tested by using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro with BAL 31 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by chloramphenicol required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role

  18. Glucomannan synthesis in pea epicotyls: the mannose and glucose transferases.

    PubMed

    Piro, G; Zuppa, A; Dalessandro, G; Northcote, D H

    1993-01-01

    Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide. PMID:7685647

  19. Characterization of prenyl protein transferase enzymes in a human keratinocyte cell line.

    PubMed

    MacNulty, E E; Ryder, N S

    1996-02-01

    Prenylation is a post-translational modification of proteins that involves the attachment of an isoprenoid group derived from mevalonic acid, either 15-carbon farnesyl or 20-carbon geranylgeranyl, to a specific carboxy-terminal domain of acceptor proteins. Three prenyl transferase enzymes have been identified so far. In this paper we report the presence of two prenyl transferases in the HaCaT human keratinocyte cell line. Chromatography of a cytosolic extract from these cells resolved a farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-I (GGPT-I) whose activities were measured using a novel peptide-based assay. Both enzymes were inhibited dose dependently by zaragozic acids A and C. Zaragozic acid C was more active towards the FPT than GGPT-I while zaragozic acid A inhibited both enzymes with similar potency. Incubation of HaCaT cell homogenates with [3H] prenyl precursors resulted in the labelling of a number of proteins which was increased when the cells were pretreated with an inhibitor of hydroxymethylglutaryl CoA reductase. Given the role of prenylated proteins in proliferative and inflammatory processes, our finding that prenyl transferases capable of prenylating endogenous substrates are also present in keratinocytes suggests that these enzymes might provide novel therapeutic targets of dermatological importance. PMID:8605230

  20. O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis

    PubMed Central

    Schimpl, Marianne; Zheng, Xiaowei; Borodkin, Vladimir S.; Blair, David E.; Ferenbach, Andrew T.; Schüttelkopf, Alexander W.; Navratilova, Iva; Aristotelous, Tonia; Albarbarawi, Osama; Robinson, David A.; Macnaughtan, Megan A.; van Aalten, Daan M.F.

    2012-01-01

    Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor. PMID:23103942

  1. The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro.

    PubMed Central

    te Heesen, S; Janetzky, B; Lehle, L; Aebi, M

    1992-01-01

    Asparagine-linked N-glycosylation is a highly conserved and functionally important modification of proteins in eukaryotic cells. The central step in this process is a cotranslational transfer of lipid-linked core oligosaccharides to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains, catalysed by the enzyme N-oligosaccharyl transferase. In this report we show that the essential yeast protein WBP1 (te Heesen et al., 1991) is required for N-oligosaccharyl transferase in vivo and in vitro. Depletion of WBP1 correlates with a defect in transferring core oligosaccharides to carboxypeptidase Y and proteinase A in vivo. In addition, in vitro N-glycosylation of the acceptor peptide Tyr-Asn-Leu-Thr-Ser-Val using microsomal membranes from WBP1 depleted cells is reduced as compared with membranes from wild-type cells. We propose that WBP1 is an essential component of the oligosaccharyl transferase in yeast. Images PMID:1600939

  2. Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

    PubMed Central

    McCaffrey, Ronald; Smoler, Donna F.; Baltimore, David

    1973-01-01

    Cells from a patient with childhood acute lymphoblastic leukemia contain an apparent DNA polymerase activity that was not found in any other cells except thymus cells. The enzyme has the properties of terminal transferase, an enzyme known to be found in thymocytes. The cells also contain the three major DNA polymerases found in growing cells. The results suggest that these tumor cells arose from a block in the differentiation of thymocytes. Terminal transferase may be a marker for the origin of leukemic cells. PMID:4346893

  3. Type II Hydride Transferases from Different Microorganisms Yield Nitrite and Diarylamines from Polynitroaromatic Compounds▿ †

    PubMed Central

    van Dillewijn, Pieter; Wittich, Rolf-Michael; Caballero, Antonio; Ramos, Juan-Luis

    2008-01-01

    Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation. PMID:18791007

  4. Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

    ERIC Educational Resources Information Center

    Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

    2011-01-01

    Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

  5. [(1)H] magnetic resonance spectroscopy of urine: diagnosis of a guanidinoacetate methyl transferase deficiency case.

    PubMed

    Tassini, Maria; Zannolli, Raffaella; Buoni, Sabrina; Engelke, Udo; Vivi, Antonio; Valensin, Gianni; Salomons, Gajja S; De Nicola, Anna; Strambi, Mirella; Monti, Lucia; Morava, Eva; Wevers, Ron A; Hayek, Joseph

    2010-01-01

    For the first time, the use of urine [(1)H] magnetic resonance spectroscopy has allowed the detection of 1 case of guanidinoacetate methyl transferase in a database sample of 1500 pediatric patients with a diagnosis of central nervous system impairment of unknown origin. The urine [(1)H] magnetic resonance spectroscopy of a 9-year-old child, having severe epilepsy and nonprogressive mental and motor retardation with no apparent cause, revealed a possible guanidinoacetic acid increase. The definitive assignment of guanidinoacetic acid was checked by addition of pure substance to the urine sample and by measuring [(1)H]-[(1)H] correlation spectroscopy. Diagnosis of guanidinoacetate methyl transferase deficiency was further confirmed by liquid chromatography-mass spectrometry, brain [(1)H] magnetic resonance spectroscopy, and mutational analysis of the guanidinoacetate methyl transferase gene. The replacement therapy was promptly started and, after 1 year, the child was seizure free. We conclude that for this case, urine [(1)H] magnetic resonance spectroscopy screening was able to diagnose guanidinoacetate methyl transferase deficiency. PMID:19461121

  6. Maize white seedling 3 results from disruption of homogentisate solanesyl transferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize white seedling 3 (w3) has served as a model albino-seedling mutant since its discovery in 1923. We show here that the w3 phenotype is caused by disruptions in homogentisate solanesyl transferase (HST), an enzyme that catalyzes the committed step in plastoquinone-9 (PQ9) biosynthesis. This re...

  7. Effect of salicylates on histamine and L-histidine metabolism. Inhibition of imidazoleacetate phosphoribosyl transferase.

    PubMed Central

    Moss, J; De Mello, M C; Vaughan, M; Beaven, M A

    1976-01-01

    In man and other animals, urinary excretion of the histidine and histamine metabolite, imidazoleacetate, is increased and that of its conjugated metabolite, ribosylimidazoleacetate, decreased by salicylates. Imidazoleacetate has been reported to produce analgesia and narcosis. Its accumulation as a result of transferase inhibition could play a part in the therapeutic effects of salicylates. To determine the locus of salicylate action, we have investigated the effect of anti-inflammatory drugs on imidazoleacetate phosphoribosyl transferase, the enzyme that catalyzes the ATP-dependent conjugation of imidazoleacetate with phosphoribosylpyrophosphate. As little as 0.2 mM aspirin produced 50% inhibition of the rat liver transferase. In vivo, a 30% decrease in the urinary excretion of ribosylimidazoleacetate has been observed with plasma salicylate concentrations of 0.4 mM. The enzyme was also inhibited by sodium salicylate but not by salicylamide, sodium gentisate, aminopyrine, phenacetin, phenylbutazone, or indomethacin. The last four drugs have been shown previously not to alter the excretion of ribosylimidazoleacetate when administered in vivo. Since both the drug specificity and inhibitory concentrations are similar in vivo and in vitro, it seems probable that the effect of salicylates on imidazoleacetate conjugation results from inhibition of imidazoleacetate phosphoribosyl transferase. PMID:180057

  8. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  9. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  10. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  11. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  12. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  13. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  14. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  15. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  16. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  17. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  18. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  19. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  20. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  1. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  2. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  3. Structural and biochemical analyses reveal how ornithine acetyl transferase binds acidic and basic amino acid substrates.

    PubMed

    Iqbal, Aman; Clifton, Ian J; Chowdhury, Rasheduzzaman; Ivison, David; Domene, Carmen; Schofield, Christopher J

    2011-09-21

    Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes. PMID:21796301

  4. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES...

  5. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  6. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  7. Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a)

    SciTech Connect

    Boone, Christopher D.; Zhong, Guo; Smeltz, Marci; James, Margaret O. McKenna, Robert

    2014-01-21

    Crystals of glutathione transferase zeta 1 were grown and shown to diffract X-rays to 3.1 Å resolution. They belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°.

  8. DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

    EPA Science Inventory


    DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

  9. GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE

    EPA Science Inventory

    ABSTRACT
    Bromodichloromethane (BDCM), a prevalent drinking water disinfection by-product, was previously shown to be mutagenic in Salmonella expressing glutathione S-transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the...

  10. A tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1).

    PubMed

    Crawford, L A; Weerapana, E

    2016-05-24

    Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition. PMID:27113843

  11. Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity.

    PubMed Central

    Gundlach, M W; Conrad, H E

    1985-01-01

    The D-glucuronosyl (GlcA)- and N-acetyl-D-galactosaminyl (GalNAc)-transferases involved in chondroitin sulphate biosynthesis were studied in a microsomal preparation from chick-embryo chondrocytes. Transfer of GlcA and GalNAc from their UDP derivatives to 3H-labelled oligosaccharides prepared from chondroitin sulphate and hyaluronic acid was assayed by h.p.l.c. of the reaction mixture. Conditions required for maximal activities of the two enzymes were remarkably similar. Activities were stimulated 3.5-6-fold by neutral detergents. Both enzymes were completely inhibited by EDTA and maximally stimulated by MnCl2 or CoCl2. MgCl2 neither stimulated nor inhibited. The GlcA transferase showed a sharp pH optimum between pH5 and 6, whereas the GalNAc transferase gave a broad optimum from pH 5 to 8. At pH 7 under optimal conditions, the GalNAc transferase gave a velocity that was twice that of the GlcA transferase. Oligosaccharides prepared from chondroitin 4-sulphate and hyaluronic acid were almost inactive as acceptors for both enzymes, whereas oligosaccharides from chondroitin 6-sulphate and chondroitin gave similar rates that were 70-80-fold higher than those observed with the endogenous acceptors. Oligosaccharide acceptors with degrees of polymerization of 6 or higher gave similar Km and Vmax. values, but the smaller oligosaccharides were less effective acceptors. These results are discussed in terms of the implications for regulation of the overall rates of the chain-elongation fractions in chondroitin sulphate synthesis in vivo. PMID:3921015

  12. Effect of glutathione S-transferase M1 polymorphisms on biomarkers of exposure and effects.

    PubMed Central

    Srám, R J

    1998-01-01

    Genotypes responsible for interindividual differences in ability to activate or detoxify genotoxic agents are recognized as biomarkers of susceptibility. Among the most studied genotypes are human glutathione transferases. The relationship of genetic susceptibility to biomarkers of exposure and effects was studied especially in relation to the genetic polymorphism of glutathione S-transferase M1 (GSTM1). For this review papers reporting the effect of GSTM1 genotype on DNA adducts, protein adducts, urine mutagenicity, Comet assay parameters, chromosomal aberrations, sister chromatid exchanges (SCE), micronuclei, and hypoxanthine-guanine phosphoribosyl transferase mutations were assessed. Subjects in groups occupationally exposed to polycyclic aromatic hydrocarbons, benzidine, pesticides, and 1,3-butadiene were included. As environmentally exposed populations, autopsy donors, coal tar-treated patients, smokers, nonsmokers, mothers, postal workers, and firefighters were followed. From all biomarkers the effect of GSTM1 and N-acetyl transferase 2 was seen in coke oven workers on mutagenicity of urine and of glutathione S-transferase T1 on the chromosomal aberrations in subjects from 1,3-butadiene monomer production units. Effects of genotypes on DNA adducts were found from lung tissue of autopsy donors and from placentas of mothers living in an air-polluted region. The GSTM1 genotype affected mutagenicity of urine in smokers and subjects from polluted regions, protein adducts in smokers, SCE in smokers and nonsmokers, and Comet assay parameters in postal workers. A review of all studies on GSTM1 polymorphisms suggests that research probably has not reached the stage where results can be interpreted to formulate preventive measures. The relationship between genotypes and biomarkers of exposure and effects may provide an important guide to the risk assessment of human exposure to mutagens and carcinogens. PMID:9539016

  13. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase.

    PubMed

    Murphy, Jesse R; Mullins, Elwood A; Kappock, T Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

  14. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase

    PubMed Central

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

  15. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

    NASA Astrophysics Data System (ADS)

    Murphy, Jesse; Mullins, Elwood; Kappock, T.

    2016-05-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates less than 3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analogue dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analogue of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

  16. Radiographic changes and lung function in relation to activity of the glutathione transferases theta and mu among asbestos cement workers.

    PubMed

    Jakobsson, K; Rannug, A; Alexandrie, A K; Warholm, M; Rylander, L; Hagmar, L

    1995-05-01

    Experimental data indicate that active oxygen species may be casually involved in the development of asbestos-related disease. Thus, it was hypothesized that individual differences in glutathione transferase activity, which may affect the ability to inactivate molecules formed in relation to oxidative stress, could influence the biological response to asbestos exposure. We could, however, not demonstrate an increased risk for radiographic changes or reduced lung function among asbestos cement workers deficient for glutathione transferase theta (GSTT1), glutathione transferase mu (GSTM1), or having a combined deficiency of enzyme activity. PMID:7618163

  17. Regiospecificity of placental metabolism by cytochromes P450 and glutathione S-transferase.

    PubMed

    McRobie, D J; Glover, D D; Tracy, T S

    1996-01-01

    The placenta possesses the ability to metabolize numerous xenobiotics and endogenous steroids. However, it is unknown whether regional differences in these enzymatic reactions exist in the human placenta. To this end, we undertook a study of four regions of the placenta, the chorionic plate, maternal surface, placental margin and whole tissue, to assess the activities of cytochrome P450 1A1 and 19A1 (aromatase) and glutathione S-stransferase in these fractions. No differences in either P450 1A1 or glutathione S-transferase activities were noted among any of the placental fractions. However, with respect to P450 19A1 activity, the placental margin differed significantly from all other fractions (p < 0.05). This study demonstrates that whole tissue samples of the human placenta are adequate for placental cytochrome P450 and glutathione S-transferase metabolism studies. PMID:8938464

  18. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription-polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  19. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori

    PubMed Central

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription–polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  20. Glutathione and gamma-glutamyl transferases are involved in the formation of cadmium-glutathione complex.

    PubMed

    Adamis, Paula Daniela Braga; Mannarino, Sérgio Cantú; Eleutherio, Elis Cristina Araújo

    2009-05-01

    In a wild-type strain of Saccharomyces cerevisiae, cadmium induces the activities of both gamma-glutamyl transferase (gamma-GT) and glutathione transferase 2 (Gtt2). However, Gtt2 activity did not increase under gamma-GT or Ycf1 deficiencies, suggesting that the accumulation of glutathione-cadmium in the cytosol inhibits Gtt2. On the other hand, the balance between the cytoplasmic and vacuolar level of glutathione seems to regulate gamma-GT activity, since this enzyme was not activated in a gtt2 strain. Taken together, these results suggest that gamma-GT and Gtt2 work together to remove cadmium from the cytoplasm, a crucial mechanism for metal detoxification that is dependent on glutathione. PMID:19345220

  1. Glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromide poisoning.

    PubMed Central

    Garnier, R; Rambourg-Schepens, M O; Müller, A; Hallier, E

    1996-01-01

    OBJECTIVES: To determine the activity of glutathione transferase and to measure the S-methylcysteine adducts in blood proteins, after acute inhalation exposure to methyl bromide. To examine the influence of the polymorphism of glutathione-S-transferase theta (GSTT1) on the neurotoxicity of methyl bromide. METHODS: Two workers acutely exposed to methyl bromide with inadequate respiratory protective devices were poisoned. Seven weeks after the accident, blood samples were drawn from both patients, for measurement of glutathione transferase activity in erythrocytes (conjugator status--that is, GSTT1 phenotype) and measurement of binding products of methyl bromide with blood proteins. Conjugator status was determined by a standard procedure. The binding product of methyl bromide, S-methylcysteine, was measured in globin and albumin. RESULTS: Duration and intensity of exposure were identical for both patients as they worked together with the same protective devices and with similar physical effort. However, one patient had very severe poisoning, whereas the other only developed mild neurotoxic symptoms. The first patient was a "conjugator" with normal glutathone transferase activity, whereas this activity was undetectable in the erythrocytes of the second patient, who consequently had higher concentrations of S-methylcysteine adduct in albumin (149 v 91 nmol/g protein) and in globin (77 v 30 nmol/g protein). CONCLUSIONS: Methyl bromide is genotoxic and neurotoxic. Its genotoxicity seems to be the consequence of the alkylating activity of the parent compound, and conjugation to glutathione has a protective effect. The data presented here suggest a different mechanism for methyl bromide neurotoxicity which could be related to the transformation of methylglutathione into toxic metabolites such as methanethiol and formaldehyde. If such metabolites are the ultimate toxic species, N-acetylcysteine treatment could have a toxifying rather than a detoxifying effect. PMID:8704864

  2. A comparison of erythrocyte glutathione S-transferase activity from human foetuses and adults.

    PubMed Central

    Strange, R C; Johnston, J D; Coghill, D R; Hume, R

    1980-01-01

    Glutathione S-transferase activity was measured in partially purified haemolysates of erythrocytes from human foetuses and adults. Enzyme activity was present in erythrocytes obtained between 12 and 40 weeks of gestation. The catalytic properties of the enzyme from foetal cells were similar to those of the enzyme from adult erythrocytes, indicating that probably only one form of the erythrocytes enzyme exists throughout foetal and adult life. PMID:7396875

  3. Homology between O-linked GlcNAc transferases and proteins of the glycogen phosphorylase superfamily.

    PubMed

    Wrabl, J O; Grishin, N V

    2001-11-30

    The O-linked GlcNAc transferases (OGTs) are a recently characterized group of largely eukaryotic enzymes that add a single beta-N-acetylglucosamine moiety to specific serine or threonine hydroxyls. In humans, this process may be part of a sugar regulation mechanism or cellular signaling pathway that is involved in many important diseases, such as diabetes, cancer, and neurodegeneration. However, no structural information about the human OGT exists, except for the identification of tetratricopeptide repeats (TPR) at the N terminus. The locations of substrate binding sites are unknown and the structural basis for this enzyme's function is not clear. Here, remote homology is reported between the OGTs and a large group of diverse sugar processing enzymes, including proteins with known structure such as glycogen phosphorylase, UDP-GlcNAc 2-epimerase, and the glycosyl transferase MurG. This relationship, in conjunction with amino acid similarity spanning the entire length of the sequence, implies that the fold of the human OGT consists of two Rossmann-like domains C-terminal to the TPR region. A conserved motif in the second Rossmann domain points to the UDP-GlcNAc donor binding site. This conclusion is supported by a combination of statistically significant PSI-BLAST hits, consensus secondary structure predictions, and a fold recognition hit to MurG. Additionally, iterative PSI-BLAST database searches reveal that proteins homologous to the OGTs form a large and diverse superfamily that is termed GPGTF (glycogen phosphorylase/glycosyl transferase). Up to one-third of the 51 functional families in the CAZY database, a glycosyl transferase classification scheme based on catalytic residue and sequence homology considerations, can be unified through this common predicted fold. GPGTF homologs constitute a substantial fraction of known proteins: 0.4% of all non-redundant sequences and about 1% of proteins in the Escherichia coli genome are found to belong to the GPGTF

  4. Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

    PubMed

    Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T

    2016-06-23

    To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats. PMID:27213958

  5. Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni.

    PubMed

    Glover, Kerney Jebrell; Weerapana, Eranthie; Numao, Shin; Imperiali, Barbara

    2005-12-01

    The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed. PMID:16356848

  6. Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida▿

    PubMed Central

    van Dillewijn, Pieter; Wittich, Rolf-Michael; Caballero, Antonio; Ramos, Juan-Luis

    2008-01-01

    To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo. PMID:18791012

  7. Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases.

    PubMed

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  8. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    PubMed Central

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  9. Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues.

    PubMed

    Buetler, T M; Eaton, D L

    1992-01-15

    Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed. PMID:1728405

  10. Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

    SciTech Connect

    Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.

    2005-01-01

    Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

  11. Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

    PubMed

    Fung, Angela Wai Shan; Ebhardt, H Alexander; Krishnakumar, Kollappillil S; Moore, Jack; Xu, Zhizhong; Strazewski, Peter; Fahlman, Richard P

    2014-07-01

    Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes. PMID:24521222

  12. Immunolabeling of Gamma-glutamyl transferase 5 in Normal Human Tissues Reveals Expression and Localization Differs from Gamma-glutamyl transferase 1

    PubMed Central

    Hanigan, Marie H.; Gillies, Elizabeth M.; Wickham, Stephanie; Wakeham, Nancy; Wirsig-Wiechmann, Celeste R.

    2014-01-01

    Gamma-glutamyl transferase (GGT5) was discovered due to its ability to convert leukotriene C4 (LTC4, a glutathione S-conjugate) to LTD4 and may have an important role in the immune system. However, it was not known which cells express the enzyme in humans. We have developed a sensitive and specific antibody that can be used to detect human GGT5 on western blots and in fixed tissue sections. We localized GGT5 expression in normal human tissues. We observed GGT5 expressed by macrophages present in many tissues, including tissue-fixed macrophages such as Kupffer cells in the liver and dust cells in the lung. GGT5 was expressed in some of the same tissues that have been shown to express gamma-glutamyl transferase (GGT1), the only other enzymatically active protein in this family. But, the two enzymes were often expressed by different cell types within the tissue. For example, GGT5 was expressed by the interstitial cells of the kidney; whereas, GGT1 is expressed on the apical surface of the renal proximal tubules. Other tissues with GGT5-positive cells included: adrenal gland, salivary gland, pituitary, thymus, spleen, liver, bone marrow, small intestine, stomach, testis, prostate and placenta. GGT5 and GGT1 are cell surface enzymes. The different pattern of expression results in their access to different extracellular fluids and therefore different substrates. GGT5 has access to substrates in blood and intercellular fluids, while GGT1 has access primarily to fluids in ducts and glands throughout the body. These data provide new insights into the different functions of these two related enzymes. PMID:25377544

  13. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  14. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis.

    PubMed

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  15. Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

    PubMed Central

    Wallrapp, Frank H.; Pan, Jian-Jung; Ramamoorthy, Gurusankar; Almonacid, Daniel E.; Hillerich, Brandan S.; Seidel, Ronald; Patskovsky, Yury; Babbitt, Patricia C.; Almo, Steven C.; Jacobson, Matthew P.; Poulter, C. Dale

    2013-01-01

    The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme–ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e−70) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally. PMID:23493556

  16. The DinB Superfamily Includes Novel Mycothiol, Bacillithiol and Glutathione S-transferases

    PubMed Central

    Newton, Gerald L.; Leung, Stephan S.; Wakabayashi, Judy I.; Rawat, Mamta; Fahey, Robert C.

    2011-01-01

    The superfamily of glutathione S-transferases has been the subject of extensive study but Actinobacteria produce mycothiol (MSH) in place of glutathione and no mycothiol S-transferase (MST) has been identified. Using mycothiol and monochlorobimane as substrates a MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily but this bacterium produces BSH. The YfiT protein was shown to have S-transferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887 but this organism produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Since more than two-thirds of the sequences assigned to the DinB superfamily are members of these families it seems likely that such activity is dominant in the DinB superfamily. PMID:22059487

  17. MIF protein are theta-class glutathione S-transferase homologs.

    PubMed Central

    Blocki, F. A.; Ellis, L. B.; Wackett, L. P.

    1993-01-01

    MIF proteins are mammalian polypeptides of approximately 13,000 molecular weight. This class includes human macrophage migration inhibitory factor (MIF), a rat liver protein that has glutathione S-transferase (GST) activity (TRANSMIF), and the mouse delayed early response gene 6 (DER6) protein. MIF proteins were previously linked to GSTs by demonstrating transferase activity and observing N-terminal sequence homology with a mu-class GST (Blocki, F.A., Schlievert, P.M., & Wackett, L.P., 1992, Nature 360, 269-270). In this study, MIF proteins are shown to be structurally related to the theta class of GSTs. This is established in three ways. First, unique primary sequence patterns are developed for each of the GST gene classes. The patterns identify the three MIF proteins as theta-like transferase homologs. Second, pattern analysis indicates that GST members of the theta class contain a serine residue in place of the N-terminal tyrosine that is implicated in glutathione deprotonation and activation in GSTs of known structure (Liu, S., et al., 1992, J. Biol. Chem. 267, 4296-4299). The MIF proteins contain a threonine at this position. Third, polyclonal antibodies raised against recombinant human MIF cross-react on Western blots with rat theta GST but not with alpha and mu GSTs. That MIF proteins have glutathione-binding ability may provide a common structural key toward understanding the varied functions of this widely distributed emerging gene family. Because theta is thought to be the most ancient evolutionary GST class, MIF proteins may have diverged early in evolution but retained a glutathione-binding domain. PMID:8298459

  18. Induction of glutathione-S-transferase activity by antioxidants in hepatocyte culture.

    PubMed

    Chen, L H; Shiau, C C

    1989-01-01

    Twelve male Sprague-Dawley rats were used for the study. Six rats were injected with benzo(a)pyrene (BP); the other six rats served as the control. Twenty-four hours after injection, hepatocytes were isolated and cultured. The cultured plates were divided into 5 groups and treated with absolute ethanol (control), butylated hydroxytoluene, vitamin E, ascorbic acid or vitamin Elascorbic acid. After 48 hours, the hepatocytes were harvested for enzyme activation determination. With both control and BP-injected rats, each antioxidant treatment significantly increased glutathione-S-transferase activity. The results suggest that antioxidants may have a detoxifying effect against BP-induced carcinogenesis. PMID:2817788

  19. The Phosphopantetheinyl Transferases: Catalysis of a Posttranslational Modification Crucial for Life

    PubMed Central

    Beld, Joris; Sonnenschein, Eva C.; Vickery, Christopher R.; Noel, Joseph P.; Burkart, Michael D.

    2014-01-01

    Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified as a distinct enzyme superfamily. In the past two decades an increasing number of papers has been published on PPTases ranging from identification, characterization, structure determination, mutagenesis, inhibition, and engineering in synthetic biology. In this review, we comprehensively discuss all current knowledge on this class of enzymes that post-translationally install a 4′-phosphopantetheine arm on various carrier proteins. PMID:24292120

  20. Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. [Pisum sativum

    SciTech Connect

    Camirand, A.; Brummell, D.; MacLachlan, G.

    1987-07-01

    Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-(/sup 14/C) fucose and UDP-(/sup 14/C)xylose to xyloglucan occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with (/sup 3/H)fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

  1. Photoactivation of hypericin down-regulates glutathione S-transferase activity in nasopharyngeal cancer cells.

    PubMed

    Du, H Y; Olivo, M; Tan, B K H; Bay, B H

    2004-04-30

    Photodynamic therapy (PDT) is a new modality of treatment for cancer. Hypericin is a photosensitizer, which is known to generate reactive oxygen species upon activation with light. We observed that photoactivated hypericin induces the generation of reactive oxygen intermediates in nasopharyngeal cancer (NPC) cells in vitro. There was also significant reduction of Glutathione S-transferase (GST) activity in HK1 and CNE-2 NPC cells and in tumor tissues from the NPC/HK1 murine tumor model by hypericin-mediated PDT. As antioxidants protect cells against phototoxicity, down-regulation of GST activity would potentiate the efficacy of hypericin-PDT treatment. PMID:15072826

  2. Glutathion S-transferase activity and DDT-susceptibility of Malaysian mosquitos.

    PubMed

    Lee, H L; Chong, W L

    1995-03-01

    Comparative DDT-susceptibility status and glutathion s-transferase (GST) activity of Malaysian Anopheles maculatus, Culex quinquefasciatus and Aedes aegypti was investigated to ascertain the role of this enzyme in DDT resistance. The standardised WHO dose-mortality bioassay tests were used to determine DDT susceptibility in these mosquitos, whilst GST microassay (Brogdon and Barber, 1990) was conducted to measure the activity of this enzyme in mosquito homogenate. It appeared that DDT susceptibility status of Malaysian mosquitos was not correlated with GST activity. PMID:8525405

  3. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity.

    PubMed

    Yates, Luke A; Durrant, Benjamin P; Fleurdépine, Sophie; Harlos, Karl; Norbury, Chris J; Gilbert, Robert J C

    2015-03-11

    Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164-N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes-for example by the binding of protein co-factors-may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules. PMID:25712096

  4. Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo.

    PubMed

    Gibbs, J B; Pompliano, D L; Mosser, S D; Rands, E; Lingham, R B; Singh, S B; Scolnick, E M; Kohl, N E; Oliff, A

    1993-04-15

    The ras oncogene product, Ras, is synthesized in vivo as a precursor protein that requires post-translational processing to become biologically active and to be capable of transforming mammalian cells. Farnesylation appears to be a critical modification of Ras, and thus inhibitors of the farnesyl-protein transferase (FPTase) that catalyzes this reaction may block ras-dependent tumorigenesis. Three structural classes of FPTase inhibitors were identified: (alpha-hydroxyfarnesyl)phosphonic acid, chaetomellic acids, and zaragozic acids. By comparison, these compounds were weaker inhibitors of geranylgeranyl-protein transferases. Each of these inhibitors was competitive with respect to farnesyl diphosphate in the FPTase reaction. All compounds were assayed for inhibition of Ras processing in Ha-ras-transformed NIH3T3 fibroblasts. Ras processing was inhibited by 1 microM (alpha-hydroxyfarnesyl)phosphonic acid. Neither chaetomellic acid nor zaragozic acid were active in this assay. These results are the first demonstration that a small organic chemical selected for inhibition of FPTase can inhibit Ras processing in vivo. PMID:8463291

  5. Structure of Human O-GlcNAc Transferase and its Complex with a Peptide Substrate

    SciTech Connect

    M Lazarus; Y Nam; J Jiang; P Sliz; S Walker

    2011-12-31

    The essential mammalian enzyme O-linked {beta}-N-acetylglucosamine transferase (O-GlcNAc transferase, here OGT) couples metabolic status to the regulation of a wide variety of cellular signalling pathways by acting as a nutrient sensor. OGT catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins, including numerous transcription factors, tumour suppressors, kinases, phosphatases and histone-modifying proteins. Aberrant glycosylation by OGT has been linked to insulin resistance, diabetic complications, cancer and neurodegenerative diseases including Alzheimer's. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 {angstrom} resolution) and as a ternary complex with UDP and a peptide substrate (1.95 {angstrom}). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT's functions; it will also help the design of inhibitors for use as cellular probes and help to assess its potential as a therapeutic target.

  6. Two Active Forms of UDP-N-Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria

    PubMed Central

    Du, Wensheng; Brown, James R.; Sylvester, Daniel R.; Huang, Jianzhong; Chalker, Alison F.; So, Chi Y.; Holmes, David J.; Payne, David J.; Wallis, Nicola G.

    2000-01-01

    Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin. PMID:10894720

  7. Selective inhibitors of glutathione transferase P1 with trioxane structure as anticancer agents.

    PubMed

    Bräutigam, Maria; Teusch, Nicole; Schenk, Tobias; Sheikh, Miriam; Aricioglu, Rocky Z; Borowski, Swantje H; Neudörfl, Jörg-Martin; Baumann, Ulrich; Griesbeck, Axel G; Pietsch, Markus

    2015-04-01

    The response to chemotherapy in cancer patients is frequently compromised by drug resistance. Although chemoresistance is a multifactorial phenomenon, many studies have demonstrated that altered drug metabolism through the expression of phase II conjugating enzymes, including glutathione transferases (GSTs), in tumor cells can be directly correlated with resistance against a wide range of marketed anticancer drugs. In particular, overexpression of glutathione transferase P1 (GSTP1) appears to be a factor for poor prognosis during cancer therapy. Former and ongoing clinical trials have confirmed GSTP1 inhibition as a principle for antitumor therapy. A new series of 1,2,4-trioxane GSTP1 inhibitors were designed via a type II photooxygenation route of allylic alcohols followed by acid-catalyzed peroxyacetalization with aldehydes. A set of novel inhibitors exhibit low micromolar to high nanomolar inhibition of GSTP1, revealing preliminary SAR for further lead optimization. Importantly, high selectivity over another two human GST classes (GSTA1 and GSTM2) has been achieved. The trioxane GSTP1 inhibitors may therefore serve as a basis for the development of novel drug candidates in overcoming chemoresistance. PMID:25694385

  8. STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo.

    PubMed Central

    Zufferey, R; Knauer, R; Burda, P; Stagljar, I; te Heesen, S; Lehle, L; Aebi, M

    1995-01-01

    N-linked glycosylation is a ubiquitous protein modification, and is essential for viability in eukaryotic cells. A lipid-linked core-oligosaccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide chains by the oligosaccharyl transferase (OTase) complex. Based on the synthetic lethal phenotype of double mutations affecting the assembly of the lipid-linked core-oligosaccharide and the OTase activity, we have performed a novel screen for mutants in Saccharomyces cerevisiae with altered N-linked glycosylation. Besides novel mutants deficient in the assembly of the lipid-linked oligosaccharide (alg mutants), we identified the STT3 locus as being required for OTase activity in vivo. The essential STT3 protein is approximately 60% identical in amino acid sequence to its human homologue. A mutation in the STT3 locus affects substrate specificity of the OTase complex in vivo and in vitro. In stt3-3 cells very little glycosyl transfer occurs from incomplete lipid-linked oligosaccharide, whereas the transfer of full-length Glc3Man9GlcNAc2 is hardly affected as compared with wild-type cells. Depletion of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex. Images PMID:7588624

  9. Structural and thermodynamic properties of kappa class glutathione transferase from Camelus dromedarius.

    PubMed

    Malik, Ajamaluddin; Fouad, Dalia; Labrou, Nikolaos E; Al-Senaidy, Abdulrahman M; Ismael, Mohamed A; Saeed, Hesham M; Ataya, Farid S

    2016-07-01

    The Arabian camel, Camelus dromedarius is naturally adapted to extreme desert climate and has evolved protective mechanisms to limit oxidative stress. The mitochondrial kappa class glutathione transferase enzyme is a member of GST supergene family that represents an important enzyme group in cellular Phase II detoxification machinery and is involved in the protection against oxidative stress and xenobiotics. In the present study, C. dromedarius kappa class glutathione transferase (CdGSTK1-1) was cloned, expressed in E. coli BL21, purified and its structural, thermodynamic and unfolding pathway was investigated. The results showed that CdGSTK1-1 has unique trimeric structure, exhibits low thermostability and a complex equilibrium unfolding profile. It unfolds through three folding states with formation of thinly populated intermediate species. The melting points (Tm) of the first unfolding transition was 40.3±0.2°C and Tm of the second unfolding transition was 49.1±0.1°C. The van't Hoff enthalpy of the first and second transition were 298.7±13.2 and 616.5±2.4kJ/mol, respectively. Moreover, intrinsic fluorescence and near-UV CD studies indicates that substrate binding does not leads to major conformational changes in CdGSTK1-1. PMID:27044344

  10. Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

    PubMed

    Kölln, Christian; Reichl, Stephan

    2016-06-15

    Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas. PMID:27113863

  11. Functional Diversification of Fungal Glutathione Transferases from the Ure2p Class

    PubMed Central

    Thuillier, Anne; Ngadin, Andrew A.; Thion, Cécile; Billard, Patrick; Jacquot, Jean-Pierre; Gelhaye, Eric; Morel, Mélanie

    2011-01-01

    The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints. PMID:22164343

  12. Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea.

    PubMed

    Vijayakumar, Harshavardhanan; Thamilarasan, Senthil Kumar; Shanmugam, Ashokraj; Natarajan, Sathishkumar; Jung, Hee-Jeong; Park, Jong-In; Kim, HyeRan; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS). Currently, understanding of their function(s) during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST) and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible) under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT) and cold susceptible (CS) lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants. PMID:27472324

  13. Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules 1

    PubMed Central

    Nelson, Oliver E.; Chourey, Prem S.; Chang, Ming Tu

    1978-01-01

    Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent Km (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10−5 M, which is compared to 3 × 10−3 M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent Km estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction. PMID:16660522

  14. Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea

    PubMed Central

    Vijayakumar, Harshavardhanan; Thamilarasan, Senthil Kumar; Shanmugam, Ashokraj; Natarajan, Sathishkumar; Jung, Hee-Jeong; Park, Jong-In; Kim, HyeRan; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS). Currently, understanding of their function(s) during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST) and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible) under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT) and cold susceptible (CS) lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants. PMID:27472324

  15. Identification of a Mycoloyl Transferase Selectively Involved in O-Acylation of Polypeptides in Corynebacteriales

    PubMed Central

    Huc, Emilie; de Sousa-D'Auria, Célia; de la Sierra-Gallay, Inès Li; Salmeron, Christophe; van Tilbeurgh, Herman; Bayan, Nicolas; Houssin, Christine

    2013-01-01

    We have previously described the posttranslational modification of pore-forming small proteins of Corynebacterium by mycolic acid, a very-long-chain α-alkyl and β-hydroxy fatty acid. Using a combination of chemical analyses and mass spectrometry, we identified the mycoloyl transferase (Myt) that catalyzes the transfer of the fatty acid residue to yield O-acylated polypeptides. Inactivation of corynomycoloyl transferase C (cg0413 [Corynebacterium glutamicum mytC {CgmytC}]), one of the six Cgmyt genes of C. glutamicum, specifically abolished the O-modification of the pore-forming proteins PorA and PorH, which is critical for their biological activity. Expectedly, complementation of the cg0413 mutant with either the wild-type gene or its orthologues from Corynebacterium diphtheriae and Rhodococcus, but not Nocardia, fully restored the O-acylation of the porins. Consistently, the three-dimensional structure of CgMytC showed the presence of a unique loop that is absent from enzymes that transfer mycoloyl residues onto both trehalose and the cell wall arabinogalactan. These data suggest the implication of this structure in the enzyme specificity for protein instead of carbohydrate. PMID:23852866

  16. Nicotinamide Mononucleotide Adenylyl Transferase 2: A Promising Diagnostic and Therapeutic Target for Colorectal Cancer

    PubMed Central

    Cui, Chunhui; Qi, Jia; Deng, Quanwen; Chen, Rihong; Zhai, Duanyang; Yu, Jinlong

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers all over the world. It is essential to search for more effective diagnostic and therapeutic methods for CRC. Abnormal nicotinamide adenine dinucleotide (NAD) metabolism has been considered as a characteristic of cancer cells. In this study, nicotinamide mononucleotide adenylyl transferases (NMNATs) as well as p53-mediated cancer signaling pathways were investigated in patients with colorectal cancer. The CRC tissues and adjacent normal tissues were obtained from 95 untreated colorectal cancer patients and were stained for expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and p53. The survival rate was analyzed by the Kaplan-Meier method and the log-rank test. The multivariate Cox proportional hazard regression analysis was conducted as well. Our data demonstrated that expression of NMNAT2 and p53 was significantly higher in CRC tissues, while NMNAT2 expression is in correlation with the invasive depth of tumors and TNM stage. Significant positive correlation was found between the expression of NMNAT2 and the expression of p53. However, NMNAT2 expression was not a statistically significant prognostic factor for overall survival. In conclusion, our results indicated that NMNAT2 might participate in tumorigenesis of CRC in a p53-dependent manner and NMNAT2 expression might be a potential therapeutic target for CRC. PMID:27218101

  17. Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target

    PubMed Central

    Dobb, Katharine S.; Kaye, Sarah J.; Beckmann, Nicola; Thain, John L.; Stateva, Lubomira; Birch, Mike; Oliver, Jason D.

    2015-01-01

    Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds. PMID:26606674

  18. Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

    SciTech Connect

    Chikanishi, Toshihiro; Fujiki, Ryoji; Hashiba, Waka; Sekine, Hiroki; Yokoyama, Atsushi; Kato, Shigeaki

    2010-04-16

    O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1{alpha} and -1{beta} genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1{alpha} gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1{alpha} gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.

  19. Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

    PubMed

    Strickland, Kyle C; Hoeferlin, L Alexis; Oleinik, Natalia V; Krupenko, Natalia I; Krupenko, Sergey A

    2010-01-15

    4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions. PMID:19933275

  20. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity

    PubMed Central

    Yates, Luke A.; Durrant, Benjamin P.; Fleurdépine, Sophie; Harlos, Karl; Norbury, Chris J.; Gilbert, Robert J.C.

    2015-01-01

    Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3′ ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164–N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes—for example by the binding of protein co-factors—may allow them alternatively to add single or multiple uridyl residues to the 3′ termini of RNA molecules. PMID:25712096

  1. Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase*

    PubMed Central

    Roston, Rebecca L.; Wang, Kun; Kuhn, Leslie A.; Benning, Christoph

    2014-01-01

    SENSITIVE TO FREEZING 2 (SFR2) is classified as a family I glycosyl hydrolase but has recently been shown to have galactosyltransferase activity in Arabidopsis thaliana. Natural occurrences of apparent glycosyl hydrolases acting as transferases are interesting from a biocatalysis standpoint, and knowledge about the interconversion can assist in engineering SFR2 in crop plants to resist freezing. To understand how SFR2 evolved into a transferase, the relationship between its structure and function are investigated by activity assay, molecular modeling, and site-directed mutagenesis. SFR2 has no detectable hydrolase activity, although its catalytic site is highly conserved with that of family 1 glycosyl hydrolases. Three regions disparate from glycosyl hydrolases are identified as required for transferase activity as follows: a loop insertion, the C-terminal peptide, and a hydrophobic patch adjacent to the catalytic site. Rationales for the effects of these regions on the SFR2 mechanism are discussed. PMID:25100720

  2. Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

    PubMed Central

    Riechers, D E; Irzyk, G P; Jones, S S; Fuerst, E P

    1997-01-01

    Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes. PMID:9276955

  3. Zaragozic acids D and D2: potent inhibitors of squalene synthase and of Ras farnesyl-protein transferase.

    PubMed

    Dufresne, C; Wilson, K E; Singh, S B; Zink, D L; Bergstrom, J D; Rew, D; Polishook, J D; Meinz, M; Huang, L; Silverman, K C

    1993-11-01

    Two new zaragozic acids, D and D2, have been isolated from the keratinophilic fungus Amauroascus niger. Zaragozic acids D [4] and D2 [5] are related to the previously described zaragozic acids A [1], B [2], and C [3] and are potent inhibitors of squalene synthase. Furthermore, all the zaragozic acids (A, B, C, D, and D2) are also active against farnesyl transferase. Zaragozic acids D and D2 inhibit farnesyl transferase with IC50 values of 100 nM, while zaragozic acids A and B are less potent. PMID:8289063

  4. Differential expression patterns of N-acetylglucosaminyl transferases and polylactosamines in uterine lesions.

    PubMed

    Clark, A T R; Guimarães da Costa, V M L; Bandeira Costa, L; Bezerra Cavalcanti, C L; De Melo Rêgo, M J B; Beltrão, E I C

    2014-01-01

    Polylactosamine (polyLacNAc) is a fundamental structure in glycoconjugates and it is expressed in specific cells/tissues associated with the development and carcinogenesis. β1,3-N-acetylglucosaminyl transferases (β3GnTs) play an important role in polyLacNAc synthesis, however the roles of these glycosyltransferases and their products in cancer progression are still unclear. In this sense, this work aimed to evaluate differential expression pattern of the N-acetylglucosaminyl transferases and polylactosamines in invasive and premalignant lesions of the uterus cervix. The expression of β3GnT2 and β3GnT3 were evaluated in normal (n=10) and uterine cervix lesions (n= 120) malignant (squamous carcinoma - SC) and premalignant (cervical intraepithelial neoplasia - CIN - grades 1, 2 and 3) using immunohistochemistry. Besides, lectin histochemistry with Phytolacca americana lectin (PWM) and Wheat germ agglutinin (WGA) was also carried out to observe the presence of polyLacNAc chains and N-acetylglucosamine (GlcNAc), respectively. The β3GnT3 was expressed in almost all samples (99%) and β3GnT2 was higher expressed in disease samples mainly in CIN 3, when compared with normal (P=0.002), CIN 1 (P=0.009) and CIN 2 (P=0.03). The expression of polyLacNAc was higher is SC samples, when compared with normal (P=0.03), CIN 1 (P=0.02) and CIN 3 (P=0.004), and was observed only nuclear expression in nearly 50% of the SC samples, showing a statistically significant when compared with normal (P=0.01), CIN 1 (P=0.002), CIN 2 (P=0.007) and CIN 3 (P=0.04). Deferring from transferases and polyLacNAc chains, GlcNAc (WGA ligand) reveals a gradual staining pattern decrease with the increase of the lesion degree, being more expressed in CIN 1 lesions when compared with normal (P<0.0001), CIN 2 (P<0.0001), SC (P<0.0001) and CIN 3 (P=0.0003). Our data reveals β3GnT2 and polyLacNAc may be involved in the progression of the pre-malignant lesions of human the uterine cervix. In addition, poly

  5. Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups

    PubMed Central

    Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

    2016-01-01

    Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs. PMID:26884677

  6. DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes.

    PubMed Central

    Sidhu, H; Ogden, S D; Lung, H Y; Luttge, B G; Baetz, A L; Peck, A B

    1997-01-01

    Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species utilizing an activation-decarboxylation reaction which yields formate and CO2. frc, the gene encoding formyl coenzyme A transferase, an enzyme which transfers a coenzyme A moiety to activate oxalic acid, was cloned from the bacterium Oxalobacter formigenes. DNA sequencing revealed a single open reading frame of 1,284 bp capable of encoding a 428-amino-acid protein. A presumed promoter region and a rho-independent termination sequence suggest that this gene is part of a monocistronic operon. A PCR fragment containing the open reading frame, when overexpressed in Escherichia coli, produced a product exhibiting enzymatic activity similar to the purified native enzyme. With this, the two genes necessary for bacterial catabolism of oxalate, frc and oxc, have now been cloned, sequenced, and expressed. PMID:9150242

  7. Rab geranylgeranyl transferase β subunit is essential for male fertility and tip growth in Arabidopsis

    PubMed Central

    Gutkowska, Malgorzata; Wnuk, Marta; Nowakowska, Julita; Lichocka, Malgorzata; Stronkowski, Michal M.; Swiezewska, Ewa

    2015-01-01

    Rab proteins, key players in vesicular transport in all eukaryotic cells, are post-translationally modified by lipid moieties. Two geranylgeranyl groups are attached to the Rab protein by the heterodimeric enzyme Rab geranylgeranyl transferase (RGT) αβ. Partial impairment in this enzyme activity in Arabidopsis, by disruption of the AtRGTB1 gene, is known to influence plant stature and disturb gravitropic and light responses. Here it is shown that mutations in each of the RGTB genes cause a tip growth defect, visible as root hair and pollen tube deformations. Moreover, FM 1–43 styryl dye endocytosis and recycling are affected in the mutant root hairs. Finally, it is demonstrated that the double mutant, with both AtRGTB genes disrupted, is non-viable due to absolute male sterility. Doubly mutated pollen is shrunken, has an abnormal exine structure, and shows strong disorganization of internal membranes, particularly of the endoplasmic reticulum system. PMID:25316062

  8. Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.

    PubMed

    Petrou, Vasileios I; Herrera, Carmen M; Schultz, Kathryn M; Clarke, Oliver B; Vendome, Jérémie; Tomasek, David; Banerjee, Surajit; Rajashankar, Kanagalaghatta R; Belcher Dufrisne, Meagan; Kloss, Brian; Kloppmann, Edda; Rost, Burkhard; Klug, Candice S; Trent, M Stephen; Shapiro, Lawrence; Mancia, Filippo

    2016-02-01

    Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes. PMID:26912703

  9. Pharmacogenetics of azathioprine in inflammatory bowel disease: a role for glutathione-S-transferase?

    PubMed

    Stocco, Gabriele; Pelin, Marco; Franca, Raffaella; De Iudicibus, Sara; Cuzzoni, Eva; Favretto, Diego; Martelossi, Stefano; Ventura, Alessandro; Decorti, Giuliana

    2014-04-01

    Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). In vivo it is active after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of isoforms M and A of the enzyme glutathione-S-transferase (GST) may increase its speed. Indeed, in pediatric patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites. In addition to increase the activation of azathioprine to mercaptopurine, GSTs may contribute to azathioprine effects even by modulating GSH consumption, oxidative stress and apoptosis. Therefore, genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed. PMID:24707136

  10. Biochemical properties of an omega-class glutathione S-transferase of the silkmoth, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Nagaoka, Sumiharu; Banno, Yutaka; Aso, Yoichi

    2009-05-01

    A cDNA encoding an omega-class glutathione S-transferase of the silkmoth, Bombyx mori (bmGSTO), was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 40, 40, and 39% identities to omega-class GSTs from human, pig, and mouse, respectively. A recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rbmGSTO was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. This enzyme was shown to have high affinity for organophosphorus insecticide and was present abundantly in silkmoth strain exhibiting fenitrothion resistance. These results indicate that bmGSTO could be involved in the increase in level of insecticide resistance for lepidopteran insects. PMID:19022397

  11. Modulating the activity of the peptidyl transferase center of the ribosome

    PubMed Central

    Beringer, Malte

    2008-01-01

    The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center. PMID:18369182

  12. Review: Human guanidinoacetate n-methyl transferase (GAMT) deficiency: A treatable inborn error of metabolism.

    PubMed

    Iqbal, Furhan

    2015-11-01

    The creatine biosynthetic pathway is essential for cellular phosphate associated energy production and storage, particularly in tissues having higher metabolic demands. Guanidinoacetate N-Methyl transferase (GAMT) is an important enzyme in creatine endogenous biosynthetic pathway, with highest expression in liver and kidney. GAMT deficiency is an inherited autosomal recessive trait that was the first among creatine deficiency syndrome to be reported in 1994 having characteristic features of no comprehensible speech development, severe mental retardation, muscular hypotonia, involuntary movements and seizures that partly cannot be treated with anti-epileptic drugs. Due to problematic endogenous creatine biosynthesis, systemic depletion of creatine/phosphocreatine and accumulation of guanidinoacetate takes place that are the diagnostic features of this disease. Dietary creatine supplementation alone or along with arginine restriction has been reported to be beneficial for all treated patients, although to various extent. However, none of the GAMT deficient patient has been reported to return to complete normal developmental level. PMID:26639513

  13. MicroRNA Regulating Glutathione S-Transferase P1 in Prostate Cancer

    PubMed Central

    Singh, Savita; Shukla, Girish C; Gupta, Sanjay

    2015-01-01

    Glutathione S-transferase P1 (GSTP1), an enzyme involved in detoxification process, is frequently inactivated in prostate cancer due to epigenetic modifications. Through in silico analysis we identified a subset of miRNAs that are putative targets in regulating GSTP1. miRNAs are small endogenous non-coding RNA that are critical regulators of various physiologic and pathologic processes and their level of expression may play a precise role in early diagnosis and prognosis of cancer. These small molecules have been detected in a wide variety of human biological specimens including blood, serum, urine, ejaculate and tissues, which could be utilized as clinically useful biomarker in early detection and prognosis of prostate cancer. The chapter summarizes the current knowledge about miRNA involved in GSTP1 regulation in prostate cancer and their potential as useful biomarkers of disease for early detection and prognosis, along with challenges and limitations in this development. PMID:25774339

  14. Single-Molecule Study of Ribosome Hierarchic Dynamics at the Peptidyl Transferase Center

    PubMed Central

    Altuntop, Mediha Esra; Ly, Cindy Tu; Wang, Yuhong

    2010-01-01

    During protein biosynthesis the ribosome moves along mRNA in steps of precisely three nucleotides. The mechanism for this ribosome motion remains elusive. Using a classification algorithm to sort single-molecule fluorescence resonance energy transfer data into subpopulations, we found that the ribosome dynamics detected at the peptidyl transferase center are highly inhomogeneous. The pretranslocation complex has at least four subpopulations that sample two hybrid states, whereas the posttranslocation complex is mainly static. We observed transitions among the ribosome subpopulations under various conditions, including 1), in the presence of EF-G; 2), spontaneously; 3), in different buffers, and 4), bound to antibiotics. Therefore, these subpopulations represent biologically active ribosomes. One key observation indicates that the Hy2 hybrid state only exists in a fluctuating ribosome subpopulation, which prompts us to propose that ribosome dynamics are hierarchically arranged. This proposal may have important implications for the regulation of cellular translation rates. PMID:21044598

  15. O-GlcNAc transferase inhibitors: current tools and future challenges.

    PubMed

    Trapannone, Riccardo; Rafie, Karim; van Aalten, Daan M F

    2016-02-01

    The O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that add and remove sugar moieties respectively. OGT knockout is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc and the dissection of catalytic compared with non-catalytic roles of OGT. Therefore, selective and potent chemical tools are necessary to inhibit OGT activity in the context of biological systems. The present review focuses on the available OGT inhibitors and summarizes advantages, limitations and future challenges. PMID:26862193

  16. Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

    SciTech Connect

    Schlunzen, Frank; Zarivach, Raz; Harms, Jörg; Bashan, Anat; Tocilj, Ante; Albrecht, Renate; Yonath, Ada; Franceschi, Francois

    2009-10-07

    Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

  17. Crystallization and preliminary X-ray analysis of glutathione transferases from cyanobacteria

    SciTech Connect

    Feil, Susanne C.; Tang, Julian; Hansen, Guido; Gorman, Michael A.; Wiktelius, Eric; Stenberg, Gun; Parker, Michael W.

    2009-05-08

    Glutathione S-transferases (GSTs) are a group of multifunctional enzymes that are found in animals, plants and microorganisms. Their primary function is to remove toxins derived from exogenous sources or the products of metabolism from the cell. Mammalian GSTs have been extensively studied, in contrast to bacterial GSTs which have received relatively scant attention. A new class of GSTs called Chi has recently been identified in cyanobacteria. Chi GSTs exhibit a high glutathionylation activity towards isothiocyanates, compounds that are normally found in plants. Here, the crystallization of two GSTs are presented: TeGST produced by Thermosynechococcus elongates BP-1 and SeGST from Synechococcus elongates PCC 6301. Both enzymes formed crystals that diffracted to high resolution and appeared to be suitable for further X-ray diffraction studies. The structures of these GSTs may shed further light on the evolution of GST catalytic activity and in particular why these enzymes possess catalytic activity towards plant antimicrobial compounds.

  18. Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase

    PubMed Central

    Aquilini, Eleonora; Merino, Susana; Knirel, Yuriy A.; Regué, Miguel; Tomás, Juan M.

    2014-01-01

    By comparison of the Proteus mirabilis HI4320 genome with known lipopolysaccharide (LPS) phosphoethanolamine transferases, three putative candidates (PMI3040, PMI3576, and PMI3104) were identified. One of them, eptC (PMI3104) was able to modify the LPS of two defined non-polar core LPS mutants of Klebsiella pneumoniae that we use as surrogate substrates. Mass spectrometry and nuclear magnetic resonance showed that eptC directs the incorporation of phosphoethanolamine to the O-6 of l-glycero-d-mano-heptose II. The eptC gene is found in all the P. mirabilis strains analyzed in this study. Putative eptC homologues were found for only two additional genera of the Enterobacteriaceae family, Photobacterium and Providencia. The data obtained in this work supports the role of the eptC (PMI3104) product in the transfer of PEtN to the O-6 of l,d-HepII in P. mirabilis strains. PMID:24756091

  19. Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein.

    PubMed

    Abdalla, Abdel-Monem; Bruns, Christopher M; Tainer, John A; Mannervik, Bengt; Stenberg, Gun

    2002-10-01

    By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages. PMID:12468717

  20. Glutathione S-transferases variants as risk factors in Alzheimer's disease.

    PubMed

    Wang, Tengfei

    2015-10-01

    Glutathione S-transferase (GST) was suggested as an important contributor to Alzheimer's disease (AD). The GSTs polymorphisms have been investigated as candidate genetic risk factors for AD, yet results remained uncertain. Therefore, we performed a meta-analysis to clarify the relationship of GSTs polymorphisms with the occurrence of AD. PubMed, Embase, Cochrane library and Alzgene databases were searched and potential literatures were selected. Pooled analyses and subgroup analyses were conducted, and also publication bias tests and cumulative meta-analysis. This meta-analysis suggested null associations between polymorphisms of GSTM1, GSTT1, GSTM3, GSTP1, GSTO1 and AD risk. GSTs variants may not have an impact on the morbidity of Alzheimer's disease. Further well designed researches are required to confirm these findings of the current study. PMID:25981226

  1. Detection and quantification of flavivirus NS5 methyl-transferase activities.

    PubMed

    Lim, Siew Pheng; Bodenreider, Christophe; Shi, Pei-Yong

    2013-01-01

    Flavivirus NS5 is the most conserved protein amongst the flavivirus proteins and is an essential enzyme for viral mRNA capping and replication. It encodes a methyl-transferase (MTase) domain at its N-terminal region which carries out sequential N7 and 2'-O methylation, resulting in the formation of the cap1 structure on its viral RNA genome. Two key methods have been established to measure these activities in vitro: thin-layer chromatography (TLC) and scintillation proximity assays (SPA). TLC offers the advantage of direct visualization of the amounts and types of cap structures formed whilst the SPA assay is more sensitive and quantitative. It is also amenable to high-throughput compound screening. The drawback of both assays is the need for radioisotope usage. We further describe the adaptation of a nonradioactive immune-competitive fluorescence polarization assay for detection of dengue virus MTase activity. PMID:23821274

  2. Terminal deoxynucleotidyl transferase in human lymphomas: possible existence of forms with high and low molecular weights.

    PubMed Central

    Vezzoni, P.; Campagnari, F.; Di Fronzo, G.; Clerici, L.

    1981-01-01

    Optimized methods for extraction and enzyme assay in crude tissue preparations were used to determine the amounts of terminal deoxnucleotidyl transferase (TdT) in malignant lymphomas. The TdT concentration was increased only in lymphoblastic lymphomas (LL) and was as high in these tumours as in the white blood cells from untreated patients with acute lymphoblastic leukaemia (ALL). The enzymes extracted from such lymphomas and from the leukaemic lymphoblasts had the same properties. Moreover, forms of TdT with low and high mol. wt were found in the LL tumours, similar to other reports of TdT-positive leukaemias. The overall study points at some basic biochemical identity of certain lymphoblastic malignancies, irrespective of whether the transformed cells are in solid tumours or are disseminated in the blood. PMID:6939447

  3. Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase.

    PubMed

    Caccuri, A M; Ascenzi, P; Antonini, G; Parker, M W; Oakley, A J; Chiessi, E; Nuccetelli, M; Battistoni, A; Bellizia, A; Ricci, G

    1996-07-01

    Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC 2.5.1.18) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2, 4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N. , Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G.(1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites. PMID:8663073

  4. Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases.

    PubMed

    Hou, Liming; Honaker, Matthew T; Shireman, Laura M; Balogh, Larissa M; Roberts, Arthur G; Ng, Kei-Cheuk; Nath, Abhinav; Atkins, William M

    2007-08-10

    The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability. PMID:17561509

  5. Kinetics and catalytic properties of coenzyme A transferase from Peptostreptococcus elsdenii.

    PubMed Central

    Schulman, M; Valentino, D

    1976-01-01

    Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii was purified to homogeneity, and some of its physical and catalytic properties were determined. The native enzyme has a molecular weight of 181,000 and is composed of two alpha subunits (molecular weight, 65,000) and one beta subunit (molecular weight 50,000). Heat treatment of the crude cell extract to 58 degrees C causes proteolysis of the native enzyme and yields a catalytically active enzyme with an approximate molecular weight of 120,000. The native CoA transferase is specific for CoA esters of short-chain alkyl monocarboxylic acids. With acetate as CoA acceptor the enzyme is active with propionyl-, butyryl-, isobutyryl-, valeryl-, isovaleryl,- and hexanoyl-CoA but not with heptanoyl or longer-chain CoA esters. There is no activity with acetoacetyl-CoA or the CoA esters of dicarboxylic acids. Steady-state kinetics indicated that the reaction proceeds via a classical bi-, bi-ping-pong mechanism. Maximal activity is obtained with propionyl- or butyryl-CoA, and both the Vmax and Km decrease as the alkyl chain length of the CoA ester increases. All CoA esters apompetitive inhibitor although it is not active as a substrate. Evidence for an enzyme CoA intermediate was provided by demonstration of an exchange between 14C-free acids (acetate and butyrate) and their corresponding CoA esters and by isolation of a 3H-labeled CoA enzyme after incubation of the enzyme with 3H-labeled acetyl-CoA. Approximately 2 mol of CoA was bound per mol of enzyme. Images PMID:977540

  6. Glutathione S-transferase GSTT1 and GSTM1 allozymes: beyond null alleles.

    PubMed

    Agúndez, José A G; Ladero, José M

    2008-03-01

    Moyer AM, Salavaggione OE, Hebbring SJ et al.: Glutathione S-transferase T1 and M1: gene sequence variation and functional genomics. Clin. Cancer Res. 13, 7207-7216 (2007). Genetic variations in the glutathione S-transferases GSTT1 and GSTM1 have been studied in many human populations, and association of these variations with environmentally-related cancers, drug-induced hepatotoxicity and even chronification of viral hepatitis has been shown. However, studies carried out to date have been limited to gene deletion, designated as null alleles, and no extensive studies on other types of genetic variations have been carried out. This study is of great importance, as it describes the occurrence and the allele frequencies for 18 SNPs in the GSTT1 gene, including four nonsynonymous SNPs, and 69 SNPs, two of which are nonsynonymous, in the GSTM1 gene. The GSTT1 SNPs leading to the amino acid substitutions Asp43Asn, Thr65Met, Thr104Pro and a single nucleotide deletion in exon 4 cause a decrease in immunoreactive protein. Interestingly, the previously described nonsynonymous GSTT1 SNPs rs2266635 (Ala21Thr), rs11550606 (Leu30Pro), rs17856199 (Phe45Cys), rs11550605 (Thr104Pro), rs2266633 (Asp141Asn) and rs2234953 (Glu173Lys) were not identified in 400 subjects, thus indicating that these variant alleles are expected to occur at extremely low frequencies. This study reinforces the need to combine SNP databases and resequencing. On combining the data reported in this study with SNP databases, the most promising target SNPs for GSTT1 association studies are those causing the amino acid changes Asp43Asn, Thr65Met, Thr104Pro and the single nucleotide deletion in exon 4. These gene variants should be analyzed in African-American and Hispanic subjects to increase the predictive capacity of genetic tests. For Caucasians and Oriental subjects, testing for null alleles seems to be sufficient. PMID:18303971

  7. Effect of three xenobiotic compounds on Glutathione S-Transferase in the clam Ruditapes decussatus.

    PubMed

    Hoarau, Pascal; Garello, Ginette; Gnassia-Barelli, Mauricette; Roméo, Michèle; Girard, Jean-Pierre

    2004-05-28

    The effects of 4,4'DDE, methoxychlor and imidazole were studied on glutathione S-transferase activities in the gills and hepatopancreas of the clam Ruditapes decussatus. The contamination doses were 0.14 microM for 4,4'DDE, 0.14 microM for methoxychlor and 25 microM for imidazole. GST activities were spectrophotometrically measured. SDS-PAGE and isoelectric focusing (IEF) were used to separate the different GST isoforms in control and treated animals, followed by Western blotting performed with anti-alpha, anti-mu and anti-pi GST anti-sera. In the hepatopancreas, GST-CDNB activities were always two to five-fold lower than in the gills where the activities were significantly increased after exposure to 4,4'DDE (ca. 1.6-fold) and to methoxychlor (ca. 1.3-fold) compared to the controls (ca. 3 micromolmin(-1)mg(-1)protein) whereas they remained unchanged after treatment with imidazole. When using glutathione S-transferase anti-alpha, anti-mu and anti-pi anti-sera, a single 26 kDa polypeptide was observed in the hepatopancreas and in the gills in all the tested conditions. Five GST subunits were observed after IEF showing greater immuno-reactivity with the anti-pi mammalian class antiserum than with the anti-alpha or anti-mu mammalian anti-sera. One isoform of pI 5.77 was particularly induced by 4,4'DDE and methoxychlor; it was recognized by the three anti-sera tested and seemed to be more efficient in the gills than in the hepatopancreas. This isoform may play a role in organochlorine detoxication. PMID:15110472

  8. ROLE OF STEROID HORMONES AND DECIDUAL INDUCTION IN THE REGULATION OF ADENOSINE DIPHOSPHORIBOSYL TRANSFERASE ACTIVITY IN RAT ENDOMETRIUM

    EPA Science Inventory

    To assess the effect of ovarian steroid hormones on enzyme activity, adenosine diphosphoribosyl transferase (ADPRT) was measured in endometrial nuclei isolated on estrus and on d 4 from rats ovariectomized on estrus (d 0) and treated d 0-3 with (a) vehicle, (b) 1 ug estrone/d (E)...

  9. Dimethyl adenosine transferase (KsgA) deficiency in Salmonella Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    : Dimethyladenosine transferase (KsgA) performs diverse roles in bacteria including ribosomal maturation, DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that ksgA mutation in Salmonella Enteritidis results in impaired invasiveness i...

  10. Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. We investigated the molecular and structural similarities between Bla g 5, a major glutathione-S transferase (GST) allergen of cockroaches, and the GST of Wucherer...

  11. Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining.

    PubMed

    Kent, Tatiana; Mateos-Gomez, Pedro A; Sfeir, Agnel; Pomerantz, Richard T

    2016-01-01

    DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3' terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3' overhangs, is facilitated by an insertion loop and conserved residues that hold the 3' primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known. PMID:27311885

  12. CHARACTERIZATION OF A p-COUMAROYL TRANSFERASE RESPONSIBLE FOR THE INCORPORATION OF pCA INTO GRASS CELL WALLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grasses form unique acylated lignins involving p-coumarate (pCA) residues primarily linked to syringyl units in lignin. A p-coumaroyl transferase (pCAT) is responsible for incorporation of pCA into cell walls as pCA-monolignol conjugates. Conjugates are synthesized in the cytoplasm, shuttled out int...

  13. The association of glutathione S-transferase polymorphisms in patients with osteosarcoma: evidence from a meta-analysis.

    PubMed

    Wang, Z; Xu, H; He, M; Wu, H; Zhu, Y; Su, Z

    2015-05-01

    Osteosarcoma is a life-threatening malignancy that often occurs in teenagers. Numerous studies have reported glutathione S-transferase polymorphisms are associated with osteosarcoma, but the results are inconclusive, partially because the sample size in each of published studies is relatively small. Therefore, we performed a meta-analysis of the published studies to estimate the association more accurately. To preciously examine the association between the glutathione S-transferase polymorphisms and osteosarcoma, we undertook a meta-analysis of six case-control studies. The association between the glutathione S-transferase polymorphisms and osteosarcoma risk was assessed by odds ratios together with their 95% confidence intervals using a fixed-effects model or random-effects model. In addition, hazard ratio was used to measure the relationship between glutathione S-transferase polymorphisms and prognosis in patients with osteosarcoma. We found that there was significant association between the polymorphisms in GSTT1 or GSTM3 (AA versus BB) and osteosarcoma risk. In addition, there is no evidence of association on GSTM1, GSTT1, GSTP1 (IIe/IIe versus IIe/Val) or GSTP1 (IIe/IIe versus Val/Val) polymorphisms with prognosis in osteosarcoma. In conclusion, the GSTT1 and GSTM3 polymorphisms might influence osteosarcoma risk. PMID:24689813

  14. Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining

    PubMed Central

    Kent, Tatiana; Mateos-Gomez, Pedro A; Sfeir, Agnel; Pomerantz, Richard T

    2016-01-01

    DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3’ terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3’ overhangs, is facilitated by an insertion loop and conserved residues that hold the 3’ primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known. DOI: http://dx.doi.org/10.7554/eLife.13740.001 PMID:27311885

  15. The TIP GROWTH DEFECTIVE1 S-Acyl Transferase Regulates Plant Cell Growth in ArabidopsisW⃞

    PubMed Central

    Hemsley, Piers A.; Kemp, Alison C.; Grierson, Claire S.

    2005-01-01

    TIP GROWTH DEFECTIVE1 (TIP1) of Arabidopsis thaliana affects cell growth throughout the plant and has a particularly strong effect on root hair growth. We have identified TIP1 by map-based cloning and complementation of the mutant phenotype. TIP1 encodes an ankyrin repeat protein with a DHHC Cys-rich domain that is expressed in roots, leaves, inflorescence stems, and floral tissue. Two homologues of TIP1 in yeast (Saccharomyces cerevisiae) and human (Homo sapiens) have been shown to have S-acyl transferase (also known as palmitoyl transferase) activity. S-acylation is a reversible hydrophobic protein modification that offers swift, flexible control of protein hydrophobicity and affects protein association with membranes, signal transduction, and vesicle trafficking within cells. We show that TIP1 binds the acyl group palmitate, that it can rescue the morphological, temperature sensitivity, and yeast casein kinase2 localization defects of the yeast S-acyl transferase mutant akr1Δ, and that inhibition of acylation in wild-type Arabidopsis roots reproduces the Tip1− mutant phenotype. Our results demonstrate that S-acylation is essential for normal plant cell growth and identify a plant S-acyl transferase, an essential research tool if we are to understand how this important, reversible lipid modification operates in plant cells. PMID:16100337

  16. BIOTRANSFORMATION AND GENOTOXICITY OF THE DRINKING WATER DISINFECTION BYPRODUCT BROMODICHLOROMETHANE: DNA BINDING MEDIATED BY GLUTATHIONE TRANSFERASE THETA 1-1

    EPA Science Inventory

    The drinking water disinfection byproduct bromodichloromethane (CHBrCl2) was
    previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione
    transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study
    to inve...

  17. LIGNIFICATION IN TRANSGENICS DEFICIENT IN P-COUMARATE 3-HYDROXYLASE (C3H) AND THE ASSOCIATED HYDROXYCINNAMOYL TRANSFERASE (HCT)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects on lignification of downregulating most of the genes for enzymes on the monolignol biosynthetic pathway have been reasonably well studied in angiosperms. The exception to this is the crucial hydroxylase, cinnamate 3-hydroxylase (C3H), and its associated hydroxycinnamyl transferase (HCT),...

  18. COMPARATIVE EXPRESSION OF TWO ALPHA CLASS GLUTATHIONE S-TRANSFERASES IN HUMAN ADULT AND PRENATAL LIVER TISSUES. (R827441)

    EPA Science Inventory

    Abstract

    The ability of the fetus to detoxify transplacental drugs and chemicals can be a critical determinant of teratogenesis and developmental toxicity. Developmentally regulated expression of alpha class glutathione S-transferases (GSTs) is of particular int...

  19. The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in humans*

    EPA Science Inventory

    Background: The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. Howev...

  20. Bilberry (Vaccinium myrtillus) Anthocyanins Modulate Heme Oxygenase-1 and Glutathione S-Transferase-pi Expression in the ARPE-19 Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PURPOSE. To determine whether anthocyanin-enriched bilberry extracts modulate pre- or post-translational levels of oxidative stress defense enzymes heme-oxygenase (HO)-1 and glutathione S-transferase-pi (GST-pi) in cultured human retinal pigment epithelial (RPE) cells. METHODS. Confluent ARPE-19 c...

  1. Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae.

    PubMed

    Naessan, Cecilia L; Egge-Jacobsen, Wolfgang; Heiniger, Ryan W; Wolfgang, Matthew C; Aas, Finn Erik; Røhr, Asmund; Winther-Larsen, Hanne C; Koomey, Michael

    2008-01-01

    The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae. PMID:17951381

  2. Modelling and bioinformatics studies of the human Kappa-class glutathione transferase predict a novel third glutathione transferase family with similarity to prokaryotic 2-hydroxychromene-2-carboxylate isomerases.

    PubMed Central

    Robinson, Anna; Huttley, Gavin A; Booth, Hilary S; Board, Philip G

    2004-01-01

    The Kappa class of GSTs (glutathione transferases) comprises soluble enzymes originally isolated from the mitochondrial matrix of rats. We have characterized a Kappa class cDNA from human breast. The cDNA is derived from a single gene comprising eight exons and seven introns located on chromosome 7q34-35. Recombinant hGSTK1-1 was expressed in Escherichia coli as a homodimer (subunit molecular mass approximately 25.5 kDa). Significant glutathione-conjugating activity was found only with the model substrate CDNB (1-chloro-2,4-ditnitrobenzene). Hyperbolic kinetics were obtained for GSH (parameters: K(m)app, 3.3+/-0.95 mM; V(max)app, 21.4+/-1.8 micromol/min per mg of enzyme), while sigmoidal kinetics were obtained for CDNB (parameters: S0.5app, 1.5+/-1.0 mM; V(max)app, 40.3+/-0.3 micromol/min per mg of enzyme; Hill coefficient, 1.3), reflecting low affinities for both substrates. Sequence analyses, homology modelling and secondary structure predictions show that hGSTK1 has (a) most similarity to bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and (b) a predicted C-terminal domain structure that is almost identical to that of bacterial disulphide-bond-forming DsbA oxidoreductase (root mean square deviation 0.5-0.6 A). The structures of hGSTK1 and HCCA isomerase are predicted to possess a thioredoxin fold with a polyhelical domain (alpha(x)) embedded between the beta-strands (betaalphabetaalpha(x)betabetaalpha, where the underlined elements represent the N and C motifs of the thioredoxin fold), as occurs in the bacterial disulphide-bond-forming oxidoreductases. This is in contrast with the cytosolic GSTs, where the helical domain occurs exclusively at the C-terminus (betaalphabetaalphabetabetaalphaalpha(x)). Although hGSTK1-1 catalyses some typical GST reactions, we propose that it is structurally distinct from other classes of cytosolic GSTs. The present study suggests that the Kappa class may have arisen in prokaryotes well before the divergence of the

  3. Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

    SciTech Connect

    Cary, J.W.; Petersen, D.J.; Bennett, G.N. ); Papoutsakis, E.T. )

    1990-06-01

    Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.

  4. Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

    SciTech Connect

    Torres, Rodrigo; Lan, Benson; Latif, Yama; Chim, Nicholas; Goulding, Celia W.

    2014-04-01

    The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NO levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

  5. Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

    SciTech Connect

    Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

    2009-02-15

    We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.

  6. Inhibition of the recombinant cattle tick Rhipicephalus (Boophilus) annulatus glutathione S-transferase.

    PubMed

    Guneidy, Rasha A; Shahein, Yasser E; Abouelella, Amira M K; Zaki, Eman R; Hamed, Ragaa R

    2014-09-01

    Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57μM compared to delphinidine-HCl (IC50=14.9±3.1μM). Gossypol had a weak inhibitory effect (IC50=43.7μM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034μM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13μM and 7.5μM, respectively. The activity of rRaGST was highest for CDNB (30.2μmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5μmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and

  7. Effects of curcumin on cytochrome P450 and glutathione S-transferase activities in rat liver.

    PubMed

    Oetari, S; Sudibyo, M; Commandeur, J N; Samhoedi, R; Vermeulen, N P

    1996-01-12

    The stability of curcumin, as well as the interactions between curcumin and cytochrome P450s (P450s) and glutathione S-transferases (GSTs) in rat liver, were studied. Curcumin is relatively unstable in phosphate buffer at pH 7.4. The stability of curcumin was strongly improved by lowering the pH or by adding glutathione (GSH), N-acetyl L-cysteine (NAC), ascorbic acid, rat liver microsomes, or rat liver cytosol. Curcumin was found to be a potent inhibitor of rat liver P450 1A1/1A2 measured as ethoxyresorufin deethylation (EROD) activity in beta-naphthoflavone (beta NF)-induced microsomes, a less potent inhibitor of P450 2B1/2B2, measured as pentoxyresorufin depentylation (PROD) activity in phenobarbital (PB)-induced microsomes and a weak inhibitor of P450 2E1, measured as p-nitrophenol (PNP) hydroxylation activity in pyrazole-induced microsomes. Ki values were 0.14 and 76.02 microM for the EROD- and PROD-activities, respectively, and 30 microM of curcumin inhibited only 9% of PNP-hydroxylation activity. In ethoxyresorufin deethylation (EROD) and pentoxyresorufin depentylation (PROD) experiments, curcumin showed a competitive type of inhibition. Curcumin was also a potent inhibitor of glutathione S-transferase (GST) activity in cytosol from liver of rats treated with phenobarbital (PB), beta-naphthoflavone (beta NF) and pyrazole (Pyr), when measured towards 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. In liver cytosol from rats treated with phenobarbital (PB), curcumin inhibited GST activity in a mixed-type manner with a Ki of 5.75 microM and Ki of 12.5 microM. In liver cytosol from rats treated with pyrazole (Pyr) or beta-naphthoflavone (beta NF), curcumin demonstrated a competitive type of inhibition with Ki values of 1.79 microM and 2.29 microM, respectively. It is concluded that these strong inhibitory properties of curcumin towards P450s and GSTs, in addition to its well-known antioxidant activity, may help explain the previously observed anticarcinogenic

  8. Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study.

    PubMed

    Comakli, Veysel; Ciftci, Mehmet; Kufrevioglu, O Irfan

    2013-12-01

    Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K(M) and Vmax values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC50 values, Ki constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag(+); Cd(2+), Cr(2+) and Mg(2+). PMID:23057421

  9. Crystal Structure of Human ADP-ribose Transferase ARTD15/PARP16 Reveals a Novel Putative Regulatory Domain*

    PubMed Central

    Karlberg, Tobias; Thorsell, Ann-Gerd; Kallas, Åsa; Schüler, Herwig

    2012-01-01

    ADP-ribosylation is involved in the regulation of DNA repair, transcription, and other processes. The 18 human ADP-ribose transferases with diphtheria toxin homology include ARTD1/PARP1, a cancer drug target. Knowledge of other family members may guide therapeutics development and help evaluate potential drug side effects. Here, we present the crystal structure of human ARTD15/PARP16, a previously uncharacterized enzyme. ARTD15 features an α-helical domain that packs against its transferase domain without making direct contact with the NAD+-binding crevice or the donor loop. Thus, this novel domain does not resemble the regulatory domain of ARTD1. ARTD15 displays auto-mono(ADP-ribosylation) activity and is affected by canonical poly(ADP-ribose) polymerase inhibitors. These results add to a framework that will facilitate research on a medically important family of enzymes. PMID:22661712

  10. Inhibition of liver glutathione S-transferase activity in rats by hypolipidemic drugs related or unrelated to clofibrate.

    PubMed

    Foliot, A; Touchard, D; Mallet, L

    1986-05-15

    The effects of in vivo administration of six hypolipidemic drugs on rat liver glutathione S-transferase activity were compared. This activity was measured with sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Except for the nicotinic acid derivative ethanolamine oxiniacate, all the compounds tested significantly reduced it, whether or not they were related to clofibrate. The hepatic glutathione concentration either remained unchanged or only increased slightly after treatment with the various drugs. When measured, the maximal excretion rate of bile BSP dropped significantly, but not that of phenol-3,6-dibromophthalein (DBSP). Hepatic dye uptake and storage were not impaired. These results show that hypolipidemic drugs of the peroxisome proliferator type inhibit rat liver glutathione S-transferase activity and may reduce transport of anions conjugated with glutathione before excretion. PMID:3707598

  11. Frequencies of glutathione s-transferase (GSTM1, GSTM3 AND GSTT1) polymorphisms in a Malaysian population

    PubMed Central

    Alshagga, Mustafa A.; Mohamed, Norazlina; Nazrun Suhid, Ahmad; Abdel Aziz Ibrahim, Ibrahim; Zulkifli Syed Zakaria, Syed

    2011-01-01

    Introduction Glutathione S-transferase (GST) is a xenobiotic metabolising enzyme (XME), which may modify susceptibility in certain ethnic groups, showing ethnic dependent polymorphism. The aim of this study was to determine GSTM1, GSTM3 and GSTT1 gene polymorphisms in a Malaysian population in Kuala Lumpur. Material and methods Blood or buccal swab samples were collected from 137 Form II students from three schools in Wilayah Persekutuan Kuala Lumpur. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results Glutathione-S-transferase GSTM3 gene frequencies were 89% for AA, 10% for AB and 1% for BB. The gene frequencies for deleted GSTM1 and GSTT1 were 66% and 18% respectively. Conclusions This study suggested that the Malay population is at risk for environmental diseases and provides the basis for gene-environment association studies to be carried out. PMID:22291790

  12. Glutathione transferase from Plasmodium falciparum--interaction with malagashanine and selected plant natural products.

    PubMed

    Mangoyi, Rumbidzai; Hayeshi, Rose; Ngadjui, Bonventure; Ngandeu, Francois; Bezabih, Merhatibebe; Abegaz, Berhanu; Razafimahefa, Solofoniaina; Rasoanaivo, Philippe; Mukanganyama, Stanley

    2010-12-01

    A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC(50) values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K(i) for GSH and CDNB were -0.015 μM and 0.011 μM, respectively. Malagashanine (100 µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t(1/2) of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5 mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds. PMID:20521884

  13. Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

    PubMed

    Cho, Ha Yeon; Maeng, Seo Jin; Cho, Hyo Je; Choi, Yoon Seo; Chung, Jeong Min; Lee, Sangmin; Kim, Hoi Kyoung; Kim, Jong Hyun; Eom, Chi-Yong; Kim, Yeon-Gil; Guo, Min; Jung, Hyun Suk; Kang, Beom Sik; Kim, Sunghoon

    2015-12-01

    Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors. PMID:26472928

  14. Transcriptomic Responses of Phanerochaete chrysosporium to Oak Acetonic Extracts: Focus on a New Glutathione Transferase

    PubMed Central

    Thuillier, Anne; Chibani, Kamel; Belli, Gemma; Herrero, Enrique; Dumarçay, Stéphane; Gérardin, Philippe; Kohler, Annegret; Deroy, Aurélie; Dhalleine, Tiphaine; Bchini, Raphael; Jacquot, Jean-Pierre; Gelhaye, Eric

    2014-01-01

    The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood. PMID:25107961

  15. Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

    PubMed Central

    Itkonen, Harri M.; Gorad, Saurabh S.; Duveau, Damien Y.; Martin, Sara E.S.; Barkovskaya, Anna; Bathen, Tone F.; Moestue, Siver A.; Mills, Ian G.

    2016-01-01

    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific. PMID:26824323

  16. A glutathione S-transferase gene associated with antioxidant properties isolated from Apis cerana cerana.

    PubMed

    Liu, Shuchang; Liu, Feng; Jia, Haihong; Yan, Yan; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2016-06-01

    Glutathione S-transferases (GSTs) are an important family of multifunctional enzymes in aerobic organisms. They play a crucial role in the detoxification of exogenous compounds, especially insecticides, and protection against oxidative stress. Most previous studies of GSTs in insects have largely focused on their role in insecticide resistance. Here, we isolated a theta class GST gene designated AccGSTT1 from Apis cerana cerana and aimed to explore its antioxidant and antibacterial attributes. Analyses of homology and phylogenetic relationships suggested that the predicted amino acid sequence of AccGSTT1 shares a high level of identity with the other hymenopteran GSTs and that it was conserved during evolution. Quantitative real-time PCR showed that AccGSTT1 is most highly expressed in adult stages and that the expression profile of this gene is significantly altered in response to various abiotic stresses. These results were confirmed using western blot analysis. Additionally, a disc diffusion assay showed that a recombinant AccGSTT1 protein may be roughly capable of inhibiting bacterial growth and that it reduces the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, these data indicate that AccGSTT1 may play an important role in antioxidant processes under adverse stress conditions. PMID:27126403

  17. Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction.

    PubMed

    Brenke, Jara K; Salmina, Elena S; Ringelstetter, Larissa; Dornauer, Scarlett; Kuzikov, Maria; Rothenaigner, Ina; Schorpp, Kenji; Giehler, Fabian; Gopalakrishnan, Jay; Kieser, Arnd; Gul, Sheraz; Tetko, Igor V; Hadian, Kamyar

    2016-07-01

    In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results. PMID:27044684

  18. Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance.

    PubMed

    Tsuboi, Katsunori; Bachovchin, Daniel A; Speers, Anna E; Spicer, Timothy P; Fernandez-Vega, Virneliz; Hodder, Peter; Rosen, Hugh; Cravatt, Benjamin F

    2011-10-19

    Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC(50) = 21 nM) and in situ (IC(50) = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance. PMID:21899313

  19. Potent and Selective Inhibitors of Glutathione S-transferase Omega 1 that Impair Cancer Drug Resistance

    PubMed Central

    Tsuboi, Katsunori; Bachovchin, Daniel A.; Speers, Anna E.; Spicer, Timothy P.; Fernandez-Vega, Virneliz; Hodder, Peter; Rosen, Hugh; Cravatt, Benjamin F.

    2011-01-01

    Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in the detoxification of chemo-therapeutic agents PMID:21899313

  20. A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis.

    PubMed

    Guan, Xin; Chen, Hui; Abramson, Alex; Man, Huimin; Wu, Jinxia; Yu, Oliver; Nikolau, Basil J

    2015-11-01

    In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo-mtACP isoforms. The mitochondrial localization of the AT3G11470-encoded proteins was validated by the ability of their N-terminal 80-residue leader sequence to guide a chimeric GFP protein to this organelle. A T-DNA-tagged null mutant mtppt-1 allele shows an embryo-lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non-photorespiratory condition (i.e. 1% CO2 atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase. PMID:26402847

  1. Solution Structure of Alg13: The Sugar Donor Subunit of a Yeast N-Acetylglucosamine Transferase

    PubMed Central

    Wang, Xu; Weldeghorghis, Thomas; Zhang, Guofeng; Imperiali, Barbara; Prestegard, James H.

    2008-01-01

    Summary The solution structure of Alg13, the glycosyl-donor binding domain of an important bipartite glycosyltransferase in the yeast S. cerevisiae, is presented. This glycosyl transferase is unusual in that it is only active in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology amongst glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B type enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and anti-parallel β sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, while conventional GT-B type enzymes usually possess three helices at the C-terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein. PMID:18547528

  2. Chlortetracycline detoxification in maize via induction of glutathione S-transferases after antibiotic exposure.

    PubMed

    Farkas, Michael H; Berry, James O; Aga, Diana S

    2007-02-15

    Soil contamination with nonmetabolized antibiotics is an emerging environmental concern, especially on agricultural croplands that receive animal manure as fertilizer. In this study, phytotoxicity of chlortetracycline (CTC) antibiotics on pinto beans (Phaseolus vulgaris) and maize (Zea mays) was investigated under controlled conditions. When grown in CTC-treated soil, a significant increase in the activities of the plant stress proteins glutathione S-transferases (GST) and peroxidases (POX) were observed in maize plants, but not in pinto beans. In vitro conjugation reactions demonstrated that the induced GST in maize catalyzed the conjugation of glutathione (GSH) with CTC, producing stable conjugates that were structurally characterized using liquid chromatography/mass spectrometry. The antibiotic-induced GST produced CTC-glutathione conjugate at relative concentrations 2-fold higher than that produced by constitutively expressed GST extracted from untreated maize. On the other hand, GST extracted from pinto beans (both treated and untreated) did not efficiently catalyze glutathione conjugation with CTC. These results suggest that maize is able to detoxify chlortetracycline via the glutathione pathway, whereas pinto beans cannot. This may explain the observed stunted growth of pinto beans after antibiotic treatment. This study demonstrates the importance of plant uptake in determining the fate of antibiotics in soil and their potential phytotoxicity to susceptible plants. PMID:17593756

  3. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria.

    PubMed

    Mills, Dominic C; Jervis, Adrian J; Abouelhadid, Sherif; Yates, Laura E; Cuccui, Jon; Linton, Dennis; Wren, Brendan W

    2016-04-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed that they were able to functionally complement the C. jejuni OTase, CjPglB. The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally, a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesized by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes. PMID:26610891

  4. Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria

    PubMed Central

    Mills, Dominic C.; Jervis, Adrian J.; Abouelhadid, Sherif; Yates, Laura E.; Cuccui, Jon; Linton, Dennis; Wren, Brendan W.

    2016-01-01

    Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed they were able to functionally complement the C. jejuni OTase, CjPglB . The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesised by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes. PMID:26610891

  5. Effects of gestational and overt diabetes on placental cytochromes P450 and glutathione S-transferase.

    PubMed

    Glover; McRobie; Tracy

    1998-07-01

    Objective: Animal and in vivo human studies have observed that diabetes alters the expression of hepatic metabolizing cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes. The placenta has the ability to metabolize a number of xenobiotic and endogenous compounds by processes similar to those seen in the liver. Our objective was to compare placental xenobiotic metabolizing activity in diabetics to matched non-diabetic controls to determine if the presence of diabetes alters placental xenobiotic metabolizing activity.Methods: The catalytic activities of 7-ethoxyresorufin-O-deethylation [EROD] (CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation with glutathione (GST) from placentas of diet controlled (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared to matched controls.Results: No differences in EROD activity were observed among overt or gestational diabetics and their respectively matched controls. CYP2E1, 2D6, and 3A4 enzyme activity were not detected in human placentas. In contrast, GST activity was significantly reduced by 30% (P <.05) in overt diabetics as compared to their matched controls and gestational diabetics.Conclusion: Pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in exposure of the fetus to harmful reactive electrophilic metabolites. PMID:10838356

  6. Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain.

    PubMed Central

    Young, P R; Briedis, A V

    1989-01-01

    The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed. PMID:2930465

  7. Effects of antioxidants on glutathione-S-transferase activities in hepatocyte culture

    SciTech Connect

    Chen, L.H. )

    1991-03-15

    Hepatocyte cultures from control rats and rats injected with 3-methylcholanthrene(3-MC) were used to study the effects of antioxidants on the activity of glutathione-S-transferases (GSH-S-T). This group of enzymes catalyzes conjugation of xenobiotics or their metabolites with reduced glutathione and plays an important role in detoxification of xenobiotics. In Experiment 1, treatment of hepatocyte cultures from both control and 3-MC-injected rats with 25 {mu}M or 50 {mu}M butylated hydroxyanisole (BHA) for 24 hours or 48 hours significantly increased GSH-S-T activity with I-chloro-2,4-dinitrobenzene (CDNB) as the substrate. In Experiment 2, treatment of hepatocytes from both control and 3-MC-treated rats with 25 {mu}M ethoxyquin or vitamin E, but not vitamin A or ascorbic acid, significantly increased GSH-S-T activity when CDNB, 1,2-dichloro-4-nitrobenzene or p-nitrobenzyl chloride was used as the substrate, respectively. The results suggested that BHA, ethoxyquin and vitamin E may have detoxification effects against 3-MC-induced carcinogenesis.

  8. The role of glutathione-S-transferase polymorphisms in ovarian cancer survival.

    PubMed

    Nagle, Christina M; Chenevix-Trench, Georgia; Spurdle, Amanda B; Webb, Penelope M

    2007-01-01

    Resistance to chemotherapy represents one of the most important causes of treatment failure in patients with ovarian cancer. Common polymorphisms in the glutathione-S-transferase (GSTM1, GSTP1 and GSTT1) family have been implicated in chemoresistence and ovarian cancer survival. In this study, we have analysed Australian women diagnosed with primary invasive epithelial ovarian cancer between 1985 and 1997, using DNA extracted from peripheral blood and archival uninvolved (normal) tissues. GSTP1 genotypes were determined using ABI Prism 7700 Sequence Detection System methodology (n=448) and GSTT1 and GSTM1 genotypes using PCR-agarose methodology (n=239). We observed a significant survival advantage among carriers of GSTP1 Ile105Val GG/GA genotype (HR 0.77, 95% confidence interval (CI) 0.61-0.99,p=0.04) and a non-significant survival advantage among women who were homozygous for the GSTM1 and GSTT1 deletion variants. There was also evidence of an additive effect, with a stronger survival benefit in women carrying three low function GST genotypes (GSTM1 null, GSTT1 null and GSTP1 GA/GG) (HR 0.47, 95% CI 0.22-1.02). The results of this study, the largest to date, are consistent with a number of previous smaller studies which have also observed that reduced GST function was associated with better survival outcomes in patients with ovarian cancer. PMID:17084623

  9. Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase.

    PubMed

    LaPensee, Elizabeth W; Schwemberger, Sandy J; LaPensee, Christopher R; Bahassi, El Mustapha; Afton, Scott E; Ben-Jonathan, Nira

    2009-08-01

    Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G(2)/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by gamma-H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTmu, but not the pi, isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options. PMID:19443905

  10. Highly ordered protein nanorings designed by accurate control of glutathione S-transferase self-assembly.

    PubMed

    Bai, Yushi; Luo, Quan; Zhang, Wei; Miao, Lu; Xu, Jiayun; Li, Hongbin; Liu, Junqiu

    2013-07-31

    Protein self-assembly into exquisite, complex, yet highly ordered architectures represents the supreme wisdom of nature. However, precise manipulation of protein self-assembly behavior in vitro is a great challenge. Here we report that by taking advantage of the cooperation of metal-ion-chelating interactions and nonspecific protein-protein interactions, we achieved accurate control of the orientation of proteins and their self-assembly into protein nanorings. As a building block, we utilized the C2-symmetric protein sjGST-2His, a variant of glutathione S-transferase from Schistosoma japonicum having two properly oriented His metal-chelating sites on the surface. Through synergic metal-coordination and non-covalent interactions, sjGST-2His self-assembled in a fixed bending manner to form highly ordered protein nanorings. The diameters of the nanorings can be regulated by tuning the strength of the non-covalent interaction network between sjGST-2His interfaces through variation of the ionic strength of the solution. This work provides a de novo design strategy that can be applied in the construction of novel protein superstructures. PMID:23865524

  11. Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

    PubMed

    Wang, Zhiling; Zhao, Zhong; Abou-Zaid, Mamdouh M; Arnason, John T; Liu, Rui; Walshe-Roussel, Brendan; Waye, Andrew; Liu, Suqi; Saleem, Ammar; Cáceres, Luis A; Wei, Qin; Scott, Ian M

    2014-12-01

    Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L. laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L. laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists. PMID:25270601

  12. Ghrelin O-Acyl Transferase in Zebrafish Is an Evolutionarily Conserved Peptide Upregulated During Calorie Restriction.

    PubMed

    Hatef, Azadeh; Yufa, Roman; Unniappan, Suraj

    2015-10-01

    Ghrelin is a multifunctional orexigenic hormone with a unique acyl modification enabled by ghrelin O-acyl transferase (GOAT). Ghrelin is well-characterized in nonmammals, and GOAT sequences of several fishes are available in the GenBank. However, endogenous GOAT in non-mammals remains poorly understood. In this research, GOAT sequence comparison, tissue-specific GOAT expression, and its regulation by nutrient status and exogenous ghrelin were studied. It was found that the bioactive core of zebrafish GOAT amino acid sequence share high identity with that of mammals. GOAT mRNA was most abundant in the gut. GOAT-like immunoreactivity (i.r.) was found colocalized with ghrelin in the gastric mucosa. Food deprivation increased, and feeding decreased GOAT and preproghrelin mRNA expression in the brain and gut. GOAT and ghrelin peptides in the gut and brain showed corresponding decrease in food-deprived state. Intraperitoneal injection of acylated fish ghrelin caused a significant decrease in GOAT mRNA expression, suggesting a feedback mechanism regulating its abundance. Together, these results provide the first in-depth characterization of GOAT in a non-mammal. Our results demonstrate that endogenous GOAT expression is responsive to metabolic status and availability of acylated ghrelin, providing further evidences for GOAT in the regulation of feeding in teleosts. PMID:26226634

  13. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal☆

    PubMed Central

    Shireman, Laura M.; Kripps, Kimberly A.; Balogh, Larissa M.; Conner, Kip P.; Whittington, Dale; Atkins, William M.

    2010-01-01

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro- 2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  14. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal.

    PubMed

    Shireman, Laura M; Kripps, Kimberly A; Balogh, Larissa M; Conner, Kip P; Whittington, Dale; Atkins, William M

    2010-12-15

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  15. Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals

    PubMed Central

    Bocedi, A; Fabrini, R; Lai, O; Alfieri, L; Roncoroni, C; Noce, A; Pedersen, JZ; Ricci, G

    2016-01-01

    Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans. PMID:27551520

  16. Inhibition of various isoforms of rat liver glutathione S-transferases by tannic acid and butein.

    PubMed

    Zhang, K; Mack, P; Wong, K P

    1997-07-01

    Glutathione S-transferases (EC.2.5.1.18, GSTs) were purified from rat liver by S-hexylglutathione affinity chromatography and six isoforms, namely C-1, C-2, C-3, C-4, A-2 and A-1, were isolated by CM-cellulose and DEAE-cellulose ion-exchange columns. Tannic acid and butein showed varying degrees of inhibition on the six individual GST isoforms. When 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate, butein exerted significantly more potent inhibition on the cationic isoforms C-2, C-3 and C-4 with IC50 values of 6.8, 8.5 and 8.0 muM respectively. All the isoforms showed lower activity towards p-nitrobenzyt chloride when compared to CDNB and inhibition of the p-nitrobenzyl chloride-activity by tannic acid and butein was also weaker. The inhibitory effects of tannic acid and butein on each isoform decreased generally with increasing pH in the range of 6.0 to 8.0. The optimum pHs for inhibitions by tannic acid and butein on the six individual isoforms lie in the pH range of 6.0 to 6.5. PMID:19856286

  17. Allyl isothiocyanate depletes glutathione and upregulates expression of glutathione S-transferases in Arabidopsis thaliana

    PubMed Central

    Øverby, Anders; Stokland, Ragni A.; Åsberg, Signe E.; Sporsheim, Bjørnar; Bones, Atle M.

    2015-01-01

    Allyl isothiocyanate (AITC) is a phytochemical associated with plant defense in plants from the Brassicaceae family. AITC has long been recognized as a countermeasure against external threats, but recent reports suggest that AITC is also involved in the onset of defense-related mechanisms such as the regulation of stomatal aperture. However, the underlying cellular modes of action in plants remain scarcely investigated. Here we report evidence of an AITC-induced depletion of glutathione (GSH) and the effect on gene expression of the detoxification enzyme family glutathione S-transferases (GSTs) in Arabidopsis thaliana. Treatment of A. thaliana wild-type with AITC resulted in a time- and dose-dependent depletion of cellular GSH. AITC-exposure of mutant lines vtc1 and pad2-1 with elevated and reduced GSH-levels, displayed enhanced and decreased AITC-tolerance, respectively. AITC-exposure also led to increased ROS-levels in the roots and loss of chlorophyll which are symptoms of oxidative stress. Following exposure to AITC, we found that GSH rapidly recovered to the same level as in the control plant, suggesting an effective route for replenishment of GSH or a rapid detoxification of AITC. Transcriptional analysis of genes encoding GSTs showed an upregulation in response to AITC. These findings demonstrate cellular effects by AITC involving a reversible depletion of the GSH-pool, induced oxidative stress, and elevated expression of GST-encoding genes. PMID:25954298

  18. Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals.

    PubMed

    Bocedi, A; Fabrini, R; Lai, O; Alfieri, L; Roncoroni, C; Noce, A; Pedersen, J Z; Ricci, G

    2016-01-01

    Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans. PMID:27551520

  19. Role of oxidative stress mediated by glutathione-s-transferase in thiopurines' toxic effects.

    PubMed

    Pelin, Marco; De Iudicibus, Sara; Fusco, Laura; Taboga, Eleonora; Pellizzari, Giulia; Lagatolla, Cristina; Martelossi, Stefano; Ventura, Alessandro; Decorti, Giuliana; Stocco, Gabriele

    2015-06-15

    Azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are antimetabolite drugs, widely used as immunosuppressants and anticancer agents. Despite their proven efficacy, a high incidence of toxic effects in patients during standard-dose therapy is recorded. The aim of this study is to explain, from a mechanistic point of view, the clinical evidence showing a significant role of glutathione-S-transferase (GST)-M1 genotype on AZA toxicity in inflammatory bowel disease patients. To this aim, the human nontumor IHH and HCEC cell lines were chosen as predictive models of the hepatic and intestinal tissues, respectively. AZA, but not 6-MP and 6-TG, induced a concentration-dependent superoxide anion production that seemed dependent on GSH depletion. N-Acetylcysteine reduced the AZA antiproliferative effect in both cell lines, and GST-M1 overexpression increased both superoxide anion production and cytotoxicity, especially in transfected HCEC cells. In this study, an in vitro model to study thiopurines' metabolism has been set up and helped us to demonstrate, for the first time, a clear role of GST-M1 in modulating AZA cytotoxicity, with a close dependency on superoxide anion production. These results provide the molecular basis to shed light on the clinical evidence suggesting a role of GST-M1 genotype in influencing the toxic effects of AZA treatment. PMID:25928802

  20. Purification and characterization of a glutathione S-transferase from Mucor mucedo.

    PubMed

    Hamed, Ragaa R; Abu-Shady, Mohamed R; El-Beih, Fawkia M; Abdalla, Abdel-Monem A; Afifi, Ola M

    2005-01-01

    An intracellular glutathione transferase was purified to homogenity from the fungus, Mucor mucedo, using DEAE-cellulose ion-exchange and glutathione affinity chromatography. Gel filtration chromatography and SDS-PAGE revealed that the purified GST is a homodimer with approximate native and subunit molecular mass of 53 kDa and 23.4 kDa, respectively. The enzyme has a pI value of 4.8, a pH optimum at pH 8.0 and apparent activation energy (Ea) of 1.42 kcal mol(-1). The purified GST acts readily on CDNB with almost negligible peroxidase activity and the activity was inhibited by Cibacron Blue (IC50 0.252 microM) and hematin (IC50 3.55 microM). M. mucedo GST displayed a non-Michaelian behavior. At low (0.1-0.3 mM) and high (0.3-2 mM) substrate concentration, Km (GSH) was calculated to be 0.179 and 0.65 mM, whereas Km(CDNB) was 0.531 and 11 mM and k(cat) was 39.8 and 552 s(-1), respectively. The enzyme showed apparent pKa values of 6-6.5 and 8.0. PMID:16209109

  1. Inhibition of human placenta glutathione transferase P1-1 by calvatic acid.

    PubMed

    Caccuri, A M; Ricci, G; Desideri, A; Buffa, M; Fruttero, R; Gasco, A; Ascenzi, P

    1994-04-01

    The inhibition mechanism of the dimeric human placenta glutathione transferase (GST P1-1) by the antibiotic p-carboxyphenylazoxycyanide (calvatic acid) has been investigated at pH 7.0 and 30.0 degrees C. Experiments performed at different calvatic acid/GST P1-1 molar ratios indicate that one mole of calvatic acid inactivates one mole of the homodimeric enzyme molecule, containing two catalytically equivalent active sites. The apparent second order rate constant for GST P1-1 inactivation is 2.4 +/- 0.3 M-1 s-1. The recovery of all the 5,5'-dithio-bis(2-nitro-benzoic acid)-titratable thiol groups as well as the original catalytic activity of GST P1-1 after treatment of the inhibited enzyme with dithiothreitol indicates that two disulfide bridges per dimer, likely between Cys47 and Cys101, have been formed during the reaction with calvatic acid. To the best of the authors knowledge, calvatic acid represents a unique case of enzyme inhibitor acting also throughout its reaction product(s). PMID:8069231

  2. Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network

    PubMed Central

    Wang, Yue-Yue; Zhang, Xiao-Sheng; Luo, Hong-Dou; Ren, Ni-Ni; Jiang, Xin-Hang; Jiang, Hui; Li, Yong-Quan

    2016-01-01

    Phosphopantetheinyl transferases (PPTases) play essential roles in both primary metabolisms and secondary metabolisms via post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). In this study, an industrial FK506 producing strain Streptomyces tsukubaensis L19, together with Streptomyces avermitilis, was identified to contain the highest number (five) of discrete PPTases known among any species thus far examined. Characterization of the five PPTases in S. tsukubaensis L19 unveiled that stw ACP, an ACP in a type II PKS, was phosphopantetheinylated by three PPTases FKPPT1, FKPPT3, and FKACPS; sts FAS ACP, the ACP in fatty acid synthase (FAS), was phosphopantetheinylated by three PPTases FKPPT2, FKPPT3, and FKACPS; TcsA-ACP, an ACP involved in FK506 biosynthesis, was phosphopantetheinylated by two PPTases FKPPT3 and FKACPS; FkbP-PCP, an PCP involved in FK506 biosynthesis, was phosphopantetheinylated by all of these five PPTases FKPPT1-4 and FKACPS. Our results here indicate that the functions of these PPTases complement each other for ACPs/PCPs substrates, suggesting a complicate phosphopantetheinylation network in S. tsukubaensis L19. Engineering of these PPTases in S. tsukubaensis L19 resulted in a mutant strain that can improve FK506 production. PMID:27052100

  3. Trichinella spiralis: low vaccine potential of glutathione S-transferase against infections in mice.

    PubMed

    Li, Ling Ge; Wang, Zhong Quan; Liu, Ruo Dan; Yang, Xuan; Liu, Li Na; Sun, Ge Ge; Jiang, Peng; Zhang, Xi; Zhang, Gong Yuan; Cui, Jing

    2015-06-01

    We have previously reported that Trichinella spiralis glutathione-S-transferase (TsGST) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML). In this study, the TsGST gene was cloned, and recombinant TsGST (rTsGST) was produced. Anti-rTsGST serum recognized the native TsGST by Western blotting in crude antigens of ML, adult worm (AW) and newborn larvae (NBL) of T. spiralis, but not in ML excretory-secretory (ES) antigens. Expression of TsGST was observed in all different developmental stages (IIL, AW, NBL and ML). An immunolocalization analysis identified TsGST in the cuticle, stichosome and genital primordium of the parasite. The rTsGST had GST enzymatic activity. After a challenge infection with T. spiralis larvae, mice immunized with rTsGST displayed a 35.71% reduction in adult worms and a 38.55% reduction in muscle larvae. The vaccination of mice with rTsGST induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1) and partial protective immunity against T. spiralis infection. PMID:25757368

  4. Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5.

    PubMed

    Brigidi, G Stefano; Santyr, Brendan; Shimell, Jordan; Jovellar, Blair; Bamji, Shernaz X

    2015-01-01

    Synaptic plasticity is mediated by the dynamic localization of proteins to and from synapses. This is controlled, in part, through activity-induced palmitoylation of synaptic proteins. Here we report that the ability of the palmitoyl-acyl transferase, DHHC5, to palmitoylate substrates in an activity-dependent manner is dependent on changes in its subcellular localization. Under basal conditions, DHHC5 is bound to PSD-95 and Fyn kinase, and is stabilized at the synaptic membrane through Fyn-mediated phosphorylation of a tyrosine residue within the endocytic motif of DHHC5. In contrast, DHHC5's substrate, δ-catenin, is highly localized to dendritic shafts, resulting in the segregation of the enzyme/substrate pair. Neuronal activity disrupts DHHC5/PSD-95/Fyn kinase complexes, enhancing DHHC5 endocytosis, its translocation to dendritic shafts and its association with δ-catenin. Following DHHC5-mediated palmitoylation of δ-catenin, DHHC5 and δ-catenin are trafficked together back into spines where δ-catenin increases cadherin stabilization and recruitment of AMPA receptors to the synaptic membrane. PMID:26334723

  5. Induction of epoxide hydrolase, glucuronosyl transferase, and sulfotransferase by phenethyl isothiocyanate in male Wistar albino rats.

    PubMed

    Abdull Razis, Ahmad Faizal; Mohd Noor, Noramaliza; Konsue, Nattaya

    2014-01-01

    Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention. PMID:24592387

  6. Identification and characterization of GSTT3, a third murine Theta class glutathione transferase.

    PubMed Central

    Coggan, Marjorie; Flanagan, Jack U; Parker, Michael W; Vichai, Vanicha; Pearson, William R; Board, Philip G

    2002-01-01

    A novel Theta class glutathione transferase (GST) isoenzyme from mouse termed mGSTT3 has been identified by analysis of the expressed sequence tag database. The gene encoding mGSTT3 is clustered with the mGSTT1 and mGSTT2 genes on chromosome 10 and has an exon/intron structure that is similar to that of the other Theta class genes. mGSTT3 is expressed strongly in the liver and to a decreasing extent in the kidney and testis. Recombinant mGSTT3-3 expressed in Escherichia coli had a substrate-specificity profile that differed significantly from that of GSTT1-1 and GSTT2-2 isoenzymes. A molecular model of mGSTT3 suggested that, in comparison with GSTT2, a decrease in volume of the hydrophobic substrate-binding site and the loss of the sulphate-binding pocket prevents its use of the GSTT2 substrate 1-menaphthyl sulphate. PMID:12038961

  7. Isothiocyanate exposure, glutathione S-transferase polymorphisms, and colorectal cancer risk1234

    PubMed Central

    Gao, Yu-Tang; Shu, Xiao-Ou; Cai, Qiuyin; Li, Guo-Liang; Li, Hong-Lan; Ji, Bu-Tian; Rothman, Nathaniel; Dyba, Marcin; Xiang, Yong-Bing; Chung, Fung-Lung; Chow, Wong-Ho; Zheng, Wei

    2010-01-01

    Background: Isothiocyanates, compounds found primarily in cruciferous vegetables, have been shown in laboratory studies to possess anticarcinogenic activity. Glutathione S-transferases (GSTs) are involved in the metabolism and elimination of isothiocyanates; thus, genetic variations in these enzymes may affect in vivo bioavailability and the activity of isothiocyanates. Objective: The objective was to prospectively evaluate the association between urinary isothiocyanate concentrations and colorectal cancer risk as well as the potential modifying effect of GST genotypes on the association. Design: A nested case-control study of 322 cases and 1251 controls identified from the Shanghai Women's Health Study was conducted. Results: Urinary isothiocyanate concentrations were inversely associated with colorectal cancer risk; the inverse association was statistically significant or nearly significant in the GSTM1-null (P for trend = 0.04) and the GSTT1-null (P for trend = 0.07) genotype groups. The strongest inverse association was found among individuals with both the GSTM1-null and the GSTT1-null genotypes, with an adjusted odds ratio of 0.51 (95% CI: 0.27, 0.95), in a comparison of the highest with the lowest tertile of urinary isothiocyanates. No apparent associations between isothiocyanate concentration and colorectal cancer risk were found among individuals who carried either the GSTM1 or GSTT1 gene (P for interaction < 0.05). Conclusion: This study suggests that isothiocyanate exposure may reduce the risk of colorectal cancer, and this protective effect may be modified by the GSTM1 and GSTT1 genes. PMID:20042523

  8. Expression profiling of selected glutathione transferase genes in Zea mays (L.) seedlings infested with cereal aphids.

    PubMed

    Sytykiewicz, Hubert; Chrzanowski, Grzegorz; Czerniewicz, Paweł; Sprawka, Iwona; Łukasik, Iwona; Goławska, Sylwia; Sempruch, Cezary

    2014-01-01

    The purpose of this report was to evaluate the expression patterns of selected glutathione transferase genes (gst1, gst18, gst23 and gst24) in the tissues of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and susceptible Tasty Sweet) that were colonized with oligophagous bird cherry-oat aphid (Rhopalosiphum padi L.) or monophagous grain aphid (Sitobion avenae L.). Simultaneously, insect-triggered generation of superoxide anion radicals (O2•-) in infested Z. mays plants was monitored. Quantified parameters were measured at 1, 2, 4, 8, 24, 48 and 72 h post-initial aphid infestation (hpi) in relation to the non-infested control seedlings. Significant increases in gst transcript amounts were recorded in aphid-stressed plants in comparison to the control seedlings. Maximal enhancement in the expression of the gst genes in aphid-attacked maize plants was found at 8 hpi (gst23) or 24 hpi (gst1, gst18 and gst24) compared to the control. Investigated Z. mays cultivars formed excessive superoxide anion radicals in response to insect treatments, and the highest overproduction of O2•- was noted 4 or 8 h after infestation, depending on the aphid treatment and maize genotype. Importantly, the Ambrozja variety could be characterized as having more profound increments in the levels of gst transcript abundance and O2•- generation in comparison with the Tasty Sweet genotype. PMID:25365518

  9. Expression Profiling of Selected Glutathione Transferase Genes in Zea mays (L.) Seedlings Infested with Cereal Aphids

    PubMed Central

    Sytykiewicz, Hubert; Chrzanowski, Grzegorz; Czerniewicz, Paweł; Sprawka, Iwona; Łukasik, Iwona; Goławska, Sylwia; Sempruch, Cezary

    2014-01-01

    The purpose of this report was to evaluate the expression patterns of selected glutathione transferase genes (gst1, gst18, gst23 and gst24) in the tissues of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and susceptible Tasty Sweet) that were colonized with oligophagous bird cherry-oat aphid (Rhopalosiphum padi L.) or monophagous grain aphid (Sitobion avenae L.). Simultaneously, insect-triggered generation of superoxide anion radicals (O2•−) in infested Z. mays plants was monitored. Quantified parameters were measured at 1, 2, 4, 8, 24, 48 and 72 h post-initial aphid infestation (hpi) in relation to the non-infested control seedlings. Significant increases in gst transcript amounts were recorded in aphid-stressed plants in comparison to the control seedlings. Maximal enhancement in the expression of the gst genes in aphid-attacked maize plants was found at 8 hpi (gst23) or 24 hpi (gst1, gst18 and gst24) compared to the control. Investigated Z. mays cultivars formed excessive superoxide anion radicals in response to insect treatments, and the highest overproduction of O2•− was noted 4 or 8 h after infestation, depending on the aphid treatment and maize genotype. Importantly, the Ambrozja variety could be characterized as having more profound increments in the levels of gst transcript abundance and O2•− generation in comparison with the Tasty Sweet genotype. PMID:25365518

  10. Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver.

    PubMed Central

    Leonard, T. B.; Neptun, D. A.; Popp, J. A.

    1984-01-01

    Serum gamma-glutamyl transferase (GGT), a marker of hepatic injury used extensively in humans, has been used rarely in rats because its specificity has not been previously defined. Studies were designed for investigation of the specificity of serum GGT activity with the use of cell type specific hepatotoxicants in Fischer 344 rats. Single necrogenic doses of CCl4, allyl alcohol (AA), and alpha-naphthylisothiocyanate (ANIT) were used to produce cell specific injury in centrilobular hepatocytes, periportal hepatocytes, and bile duct cells, respectively. Administration of CCl4 markedly increased serum activities of alanine aminotransferase (ALT), alkaline phosphatase (AP), and serum bile acid concentrations within 24 hours but had no effect on serum GGT activity. ANIT treatment increased serum GGT and AP activities and bile acid concentration 24 hours following administration. Allyl alcohol administration increased serum ALT activity but had no effect on GGT activity. Administration of ANIT in the diet at 0.01%, 0.022%, 0.047%, and 0.1% for 2, 4, and 6 weeks produced dose- and time-dependent increases in serum GGT activity which strongly correlated with quantitative increases in hepatic bile duct volume, which was determined morphometrically. These observations support the use of serum GGT activity in the rat as diagnostic of bile duct cell necrosis when increases are detected shortly after the insult and as an indicator of possible bile duct hyperplasia. Images Figure 1 Figure 3 PMID:6147091

  11. Serum gamma glutamyl transferase as a specific indicator of bile duct lesions in the rat liver.

    PubMed

    Leonard, T B; Neptun, D A; Popp, J A

    1984-08-01

    Serum gamma-glutamyl transferase (GGT), a marker of hepatic injury used extensively in humans, has been used rarely in rats because its specificity has not been previously defined. Studies were designed for investigation of the specificity of serum GGT activity with the use of cell type specific hepatotoxicants in Fischer 344 rats. Single necrogenic doses of CCl4, allyl alcohol (AA), and alpha-naphthylisothiocyanate (ANIT) were used to produce cell specific injury in centrilobular hepatocytes, periportal hepatocytes, and bile duct cells, respectively. Administration of CCl4 markedly increased serum activities of alanine aminotransferase (ALT), alkaline phosphatase (AP), and serum bile acid concentrations within 24 hours but had no effect on serum GGT activity. ANIT treatment increased serum GGT and AP activities and bile acid concentration 24 hours following administration. Allyl alcohol administration increased serum ALT activity but had no effect on GGT activity. Administration of ANIT in the diet at 0.01%, 0.022%, 0.047%, and 0.1% for 2, 4, and 6 weeks produced dose- and time-dependent increases in serum GGT activity which strongly correlated with quantitative increases in hepatic bile duct volume, which was determined morphometrically. These observations support the use of serum GGT activity in the rat as diagnostic of bile duct cell necrosis when increases are detected shortly after the insult and as an indicator of possible bile duct hyperplasia. PMID:6147091

  12. A glutathione S-transferase gene associated with antioxidant properties isolated from Apis cerana cerana

    NASA Astrophysics Data System (ADS)

    Liu, Shuchang; Liu, Feng; Jia, Haihong; Yan, Yan; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2016-06-01

    Glutathione S-transferases (GSTs) are an important family of multifunctional enzymes in aerobic organisms. They play a crucial role in the detoxification of exogenous compounds, especially insecticides, and protection against oxidative stress. Most previous studies of GSTs in insects have largely focused on their role in insecticide resistance. Here, we isolated a theta class GST gene designated AccGSTT1 from Apis cerana cerana and aimed to explore its antioxidant and antibacterial attributes. Analyses of homology and phylogenetic relationships suggested that the predicted amino acid sequence of AccGSTT1 shares a high level of identity with the other hymenopteran GSTs and that it was conserved during evolution. Quantitative real-time PCR showed that AccGSTT1 is most highly expressed in adult stages and that the expression profile of this gene is significantly altered in response to various abiotic stresses. These results were confirmed using western blot analysis. Additionally, a disc diffusion assay showed that a recombinant AccGSTT1 protein may be roughly capable of inhibiting bacterial growth and that it reduces the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, these data indicate that AccGSTT1 may play an important role in antioxidant processes under adverse stress conditions.

  13. Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

    SciTech Connect

    Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling; Gorman, Michael A.; Morton, Craig J.; Pyke, James; McConville, Malcolm J.; Bieri, Michael; Mok, Yee-Foong; Robin, Charles; Gooley, Paul R.; Parker, Michael W.; Batterham, Philip

    2010-06-14

    GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.

  14. Septins guide microtubule protrusions induced by actin-depolymerizing toxins like Clostridium difficile transferase (CDT).

    PubMed

    Nölke, Thilo; Schwan, Carsten; Lehmann, Friederike; Østevold, Kristine; Pertz, Olivier; Aktories, Klaus

    2016-07-12

    Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton. PMID:27339141

  15. Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases

    PubMed Central

    Henderson, Colin J.; Smith, Austin G.; Ure, Jan; Brown, Ken; Bacon, E. Jane; Wolf, C. Roland

    1998-01-01

    The activity of chemical carcinogens is a complex balance between metabolic activation by cytochrome P450 monooxygenases and detoxification by enzymes such as glutathione S-transferase (GST). Regulation of these proteins may have profound effects on carcinogenic activity, although it has proved impossible to ascribe the observed effects to the activity of a single protein. GstP appears to play a very important role in carcinogenesis, although the precise nature of its involvement is unclear. We have deleted the murine GstP gene cluster and established the effects on skin tumorigenesis induced by the polycyclic aromatic hydrocarbon 7,12-dimethylbenz anthracene and the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate. After 20 weeks, a highly significant increase in the number of papillomas was found in the GstP1/P2 null mice [GstP1/P2(−/−) mice, 179 papillomas, mean 9.94 per animal vs. GstP1/P2(+/+) mice, 55 papillomas, mean 2.89 per animal, (P < 0.001)]. This difference in tumor incidence provides direct evidence that a single gene involved in drug metabolism can have a profound effect on tumorigenicity, and demonstrates that GstP may be an important determinant in cancer susceptibility, particularly in diseases where exposure to polycyclic aromatic hydrocarbons is involved, for instance in cigarette smoke-induced lung cancer. PMID:9560266

  16. Dual functionality of O-GlcNAc transferase is required for Drosophila development.

    PubMed

    Mariappa, Daniel; Zheng, Xiaowei; Schimpl, Marianne; Raimi, Olawale; Ferenbach, Andrew T; Müller, H-Arno J; van Aalten, Daan M F

    2015-12-01

    Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein-protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity. PMID:26674417

  17. Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

    SciTech Connect

    Dehennaut, Vanessa; Hanoulle, Xavier; Bodart, Jean-Francois; Vilain, Jean-Pierre; Michalski, Jean-Claude; Landrieu, Isabelle; Lippens, Guy; Lefebvre, Tony

    2008-05-02

    In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads to a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.

  18. The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

    PubMed

    Levine, Zebulon G; Walker, Suzanne

    2016-06-01

    O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding β-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells. PMID:27294441

  19. Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic {beta} cells

    SciTech Connect

    Andrali, Sreenath S.; Maerz, Pia; Oezcan, Sabire . E-mail: sozcan@uky.edu

    2005-11-11

    Several nuclear and cytoplasmic proteins in metazoans are modified by O-linked N-acetylglucosamine (O-GlcNAc). This modification is dynamic and reversible similar to phosphorylation and is catalyzed by the O-linked GlcNAc transferase (OGT). Hyperglycemia has been shown to increase O-GlcNAc levels in pancreatic {beta} cells, which appears to interfere with {beta}-cell function. To obtain a better understanding of the role of O-linked GlcNAc modification in {beta} cells, we have isolated OGT interacting proteins from a cDNA library made from the mouse insulinoma MIN6 cell line. We describe here the identification of Ataxin-10, encoded by the SCA10 (spinocerebellar ataxia type 10) gene as an OGT interacting protein. Mutations in the SCA10 gene cause progressive cerebellar ataxias and seizures. We demonstrate that SCA10 interacts with OGT in vivo and is modified by O-linked glycosylation in MIN6 cells, suggesting a novel role for the Ataxin-10 protein in pancreatic {beta} cells.

  20. Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism.

    PubMed

    Itkonen, Harri M; Gorad, Saurabh S; Duveau, Damien Y; Martin, Sara E S; Barkovskaya, Anna; Bathen, Tone F; Moestue, Siver A; Mills, Ian G

    2016-03-15

    Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific. PMID:26824323

  1. Glutathione S-transferase polymorphisms in varicocele patients: a meta-analysis.

    PubMed

    Zhu, B; Yin, L; Zhang, J Y

    2015-01-01

    The glutathione S-transferase (GST) family represents a major group of detoxification and antioxidant enzymes. Studies have shown that high oxidative stress levels are associated with varicocele. The objective of this study was to assess the relationship between GSTM1 and GSTT1 null polymorphisms and varicocele using a study group of 497 varicocele patients and 476 control subjects. A systematic literature search (for articles published up to September 2014) utilizing Google Scholar and PubMed was conducted. The chi-square-based Q test and I(2) index were used to evaluate data from retrieved studies. The possible publication bias was evaluated by Begg funnel plot and the Egger test. No statistically significant association was found between GSTM1 or GSTT1 null genotypes and varicocele in the overall data analysis. In a subgroup analysis, only the null GSTM1 genotype was observed at a significantly higher frequency in Caucasian varicocele patients. In the Chinese subgroup, no association was established between the GSTM1 and GSTT1 null genotypes and this condition. More attention should be drawn to oxidative stress-related pathological manifestations for Caucasian varicocele patients. PMID:26782535

  2. Protein S-ACYL Transferase10 is critical for development and salt tolerance in Arabidopsis.

    PubMed

    Zhou, Liang-Zi; Li, Sha; Feng, Qiang-Nan; Zhang, Yu-Ling; Zhao, Xinying; Zeng, Yong-lun; Wang, Hao; Jiang, Liwen; Zhang, Yan

    2013-03-01

    Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B-like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins. PMID:23482856

  3. Functional Analyses of Mycobacterial Lipoprotein Diacylglyceryl Transferase and Comparative Secretome Analysis of a Mycobacterial lgt Mutant

    PubMed Central

    Tschumi, Andreas; Grau, Thomas; Albrecht, Dirk; Rezwan, Mandana; Antelmann, Haike

    2012-01-01

    Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and [14C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target. PMID:22609911

  4. Fluorometric microplate assay to measure glutathione S-transferase activity in insects and mites using monochlorobimane.

    PubMed

    Nauen, Ralf; Stumpf, Natascha

    2002-04-15

    Elevated levels of glutathione S-transferases (GSTs) play a major role as a mechanism of resistance to insecticides and acaricides in resistant pest insects and mites, respectively. Such compounds are either detoxicated directly via phase I metabolism or detoxicated by phase II metabolism of metabolites as formed by microsomal monooxygenases. Here we used monochlorobimane (MCB) as an artificial substrate and glutathione to determine total GST activity in equivalents of single pest insects and spider mites in a sensitive 96-well plate-based assay system by measuring the enzymatic conversion of MCB to its fluorescent bimane-glutathione adduct. The differentiation by their GST activity between several strains of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), with different degrees of resistance to numerous acaricides was more sensitive with MCB compared to the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Compared to an acaricide-susceptible reference strain, one field population of T. urticae showed a more than 10-fold higher GST activity measured with MCB, in contrast to a less than 2-fold higher activity when CDNB was used. Furthermore, we showed that GST activity can be sensitively assessed with MCB in homogenates of pest insects such as Heliothis virescens, Spodoptera frugiperda (Lepidoptera: Noctuidae), Plutella xylostella (Lepidoptera: Yponomeutidae), and Myzus persicae (Hemiptera: Aphididae). PMID:11950219

  5. Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.

    PubMed Central

    Neufeld, K L; Galarza, J M; Richards, O C; Summers, D F; Ehrenfeld, E

    1994-01-01

    A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA. Images PMID:8057462

  6. Staphylococcus aureus formyl-methionyl transferase mutants demonstrate reduced virulence factor production and pathogenicity.

    PubMed

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; Demarsh, Peter; Aubart, Kelly; Zalacain, Magdalena

    2013-07-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  7. Glutathione S-transferases of Aulacorthum solani and Acyrthosiphon pisum: partial purification and characterization.

    PubMed

    Francis, F; Haubruge, E; Gaspar, C; Dierickx, P J

    2001-05-01

    Glutathione S-transferases (GST) play an important role in the detoxification of many substances including allelochemicals from plants. Brassicaceae plants contain glucosinolates and emit volatile isothiocyanates which affect the GST system. A comparison of the GST of two aphid species, the generalist Aulacorthum solani found on Brassicaceae and the Fabaceae specialist Acyrthosiphon pisum, was made to try to explain their respective feeding behaviour. Differences of GST were determined among the two aphid species based on purification by affinity chromatography, SDS-PAGE and on kinetic studies. Purification yields using an epoxy-activated Sepharose 6B column were highly different for the two aphid species (18% and 34% for A. solani and A. pisum, respectively). These variations were confirmed by SDS-PAGE. While only a 27-kDa band was observed for A. pisum, two bands of approximately 25-kDa were visualized for the generalist aphid, A. solani. Considering the kinetic results, differences of Km and Vmax were observed following the aphid species when a range of substrates (CDNB and DCNB) and GSH concentrations were tested. Studies on the detoxification enzymes of generalist and specialist herbivores would be undertaken to determine accurately the effect of the host plant on the organisms eating them, particularly in terms of biochemical and ecological advantages. PMID:11337260

  8. Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.

    PubMed

    Guengerich, F Peter; Wheeler, James B; Chun, Young-Jin; Kim, Donghak; Shimada, Tsutomu; Aryal, Pramod; Oda, Yoshimitsu; Gillam, Elizabeth M J

    2002-12-27

    Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. PMID:12505322

  9. Optical biosensor consisting of glutathione-S-transferase for detection of captan.

    PubMed

    Choi, Jeong-Woo; Kim, Young-Kee; Song, Sun-Young; Lee, In-ho; Lee, Won-Hong

    2003-10-15

    The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 microg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min. PMID:12941561

  10. Glutathione-S-Transferases As Determinants of Cell Survival and Death

    PubMed Central

    Townsend, Danyelle M.

    2012-01-01

    Abstract Significance: The family of glutathione S-transferases (GSTs) is part of a cellular Phase II detoxification program composed of multiple isozymes with functional human polymorphisms that have the capacity to influence individual response to drugs and environmental stresses. Catalytic activity is expressed through GST dimer-mediated thioether conjugate formation with resultant detoxification of a variety of small molecule electrophiles. Recent Advances: More recent work indicates that in addition to the classic catalytic functions, specific GST isozymes have other characteristics that impact cell survival pathways in ways unrelated to detoxification. These characteristics include the following: regulation of mitogen-activated protein kinases; facilitation of the addition of glutathione to cysteine residues in certain proteins (S-glutathionylation); as a novel cellular partner of the human papilloma virus-16 E7 oncoprotein playing a pivotal role in preventing cell death in infected human cells; mitogenic influence in myeloproliferative pathways; participant in the process of cocaine addiction. Critical Issues: Some of these functions have provided a platform for targeting GST with novel small molecule therapeutics, particularly in cancer where evidence of clinical applications is emerging. Future Directions: Our evolving understanding of the GST superfamily and their divergent expression patterns in individuals make them attractive candidates for translational studies in a variety of human pathologies. In addition, their role in regulating cell fate in signaling and cell death pathways has opened up a significant functional complexity that extends well beyond standard detoxification reactions. Antioxid. Redox Signal. 17, 1728–1737. PMID:22540427

  11. Association between herbivore stress and glutathione S-transferase expression in Pinus brutia Ten.

    PubMed

    Semiz, A; Çelik-Turgut, G; Semiz, G; Özgün, Ö; Şen, A

    2016-01-01

    Plants have developed mechanisms to defend themselves against many factors including biotic stress such as herbivores and pathogens. Glutathione S-transferase (GST) is a glutathione-dependent detoxifying enzyme and plays critical roles in stress tolerance and detoxification metabolism in plants. Pinus brutia Ten. is a prominent native forest tree species in Turkey, due to both its economic and ecological assets. One of the problems faced by P. brutia afforestation sites is the attacks by pine processionary moth (Thaumetopoea wilkinsoni Tams.). In this study, we investigated the changes in activity and mRNA expression of GST in pine samples taken from both resistant and susceptible clones against T. wilkinsoni over a nine month period in a clonal seed orchard. It was found that the average cytosolic GST activities of trees in March and July were significantly higher than the values obtained in November. November was considered to be the control since trees were not under stress yet. In addition, RT-PCR results clearly showed that levels of GST transcripts in March and July samples were significantly higher as compared to the level seen in November. These findings strongly suggest that GST activity from P. brutia would be a valuable marker for exposure to herbivory stress. PMID:27064879

  12. Comprehensive X-Ray Structural Studies of the Quinolinate Phosphoribosyl Transferase (BNA6) From Saccharomyces Cerevisiae

    SciTech Connect

    di Luccio, E.; Wilson, D.K.

    2009-05-14

    Quinolinic acid phosphoribosyl transferase (QAPRTase, EC 2.4.2.19) is a 32 kDa enzyme encoded by the BNA6 gene in yeast and catalyzes the formation of nicotinate mononucleotide from quinolinate and 5-phosphoribosyl-1-pyrophosphate (PRPP). QAPRTase plays a key role in the tryptophan degradation pathway via kynurenine, leading to the de novo biosynthesis of NAD{sup +} and clearing the neurotoxin quinolinate. To improve our understanding of the specificity of the eukaryotic enzyme and the course of events associated with catalysis, we have determined the crystal structures of the apo and singly bound forms with the substrates quinolinate and PRPP. This reveals that the enzyme folds in a manner similar to that of various prokaryotic forms which are {approx}30% identical in sequence. In addition, the structure of the Michaelis complex is approximated by PRPP and the quinolinate analogue phthalate bound to the active site. These results allow insight into the kinetic mechanism of QAPRTase and provide an understanding of structural diversity in the active site of the Saccharomyces cerevisiae enzyme when compared to prokaryotic homologues.

  13. Discovery of a novel nicotinamide phosphoribosyl transferase (NAMPT) inhibitor via in silico screening.

    PubMed

    Takeuchi, Mikio; Niimi, Tatsuya; Masumoto, Mari; Orita, Masaya; Yokota, Hiroyuki; Yamamoto, Tomoko

    2014-01-01

    Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC50 of 44 nM, and inhibited THP-1 and K562 cell line growth with the IC50 of 198 nM and 673 nM, respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT. PMID:24389478

  14. Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

    PubMed Central

    Lewandowski, Thomas; Huang, Jianzhong; Fan, Frank; Rogers, Shannon; Gentry, Daniel; Holland, Reannon; DeMarsh, Peter; Zalacain, Magdalena

    2013-01-01

    Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. PMID:23571548

  15. Role of glutathione S-transferases in the spinocerebellar ataxia type 2 clinical phenotype.

    PubMed

    Almaguer-Gotay, D; Almaguer-Mederos, L E; Aguilera-Rodríguez, R; Estupiñán-Rodríguez, A; González-Zaldivar, Y; Cuello-Almarales, D; Laffita-Mesa, J M; Vázquez-Mojena, Y

    2014-06-15

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative and incurable hereditary disorder caused by a CAG repeat expansion mutation on ATXN2 gene. The identification of reliable biochemical markers of disease severity is of paramount significance for the development and assessment of clinical trials. In order to evaluate the potential use of glutathione-S-transferase (GST) activity as a biomarker for SCA2, a case-control study in 38 affected, presymptomatic individuals or healthy controls was conducted. An enlarged sample of 121 affected individuals was set to assess the impact of GST activity on SCA2 clinical expression. There was a significant increase in GST activity in affected individuals relative to controls, although sensibility and specificity were not high. GST activity was not significantly influenced by sex, age, disease duration or CAG repeat size and did not significantly influence disease severity markers. These findings show a disruption of in vivo GST activity in SCA2, suggesting a role for oxidative stress in the neurodegenerative process. PMID:24780439

  16. The Impact of Nitric Oxide Toxicity on the Evolution of the Glutathione Transferase Superfamily

    PubMed Central

    Bocedi, Alessio; Fabrini, Raffaele; Farrotti, Andrea; Stella, Lorenzo; Ketterman, Albert J.; Pedersen, Jens Z.; Allocati, Nerino; Lau, Peter C. K.; Grosse, Stephan; Eltis, Lindsay D.; Ruzzini, Antonio; Edwards, Thomas E.; Morici, Laura; Del Grosso, Erica; Guidoni, Leonardo; Bovi, Daniele; Lo Bello, Mario; Federici, Giorgio; Parker, Michael W.; Board, Philip G.; Ricci, Giorgio

    2013-01-01

    Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10−9 m) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 102–104 times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO. PMID:23828197

  17. The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP

    PubMed Central

    Wurm, Torsten; Wright, Diana G.; Polakowski, Nicholas; Mesnard, Jean-Michel; Lemasson, Isabelle

    2012-01-01

    The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection. PMID:22434882

  18. The active site of O-GlcNAc transferase imposes constraints on substrate sequence

    PubMed Central

    Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

    2016-01-01

    O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

  19. Association study of Glutathione S-Transferase polymorphisms and risk of endometriosis in an Iranian population

    PubMed Central

    Hassani, Mina; Saliminejad, Kioomars; Heidarizadeh, Masood; Kamali, Koorosh; Memariani, Toktam; Khorram Khorshid, Hamid Reza

    2016-01-01

    Background: Endometriosis influenced by both genetic and environmental factors. Associations of glutathione S-transferases (GSTs) genes polymorphisms in endometriosis have been investigated by various researchers; however, the results are not consistent. Objective: We examined the associations of GSTM1 and GSTT1 null genotypes and GSTP1 313 A/G polymorphisms with endometriosis in an Iranian population. Materials and Methods: In this case-control study, 151 women with diagnosis of endometriosis and 156 normal healthy women as control group were included. The genotyping was determined using multiplex PCR and PCR- RFLP methods. Results: The GSTM1 null genotype was significantly higher (p=0.027) in the cases (7.3%) than the control group (1.3%). There was no significant difference between the frequency of GSTT1 genotypes between the cases and controls. The GSTP1 313 AG genotype was significantly lower (p=0.048) in the case (33.1%) than the control group (44.4%). Conclusion: Our results showed that GSTM1 and GSTP1 polymorphisms may be associated with susceptibility of endometriosis in Iranian women. PMID:27351025

  20. Dual functionality of O-GlcNAc transferase is required for Drosophila development

    PubMed Central

    Mariappa, Daniel; Zheng, Xiaowei; Schimpl, Marianne; Raimi, Olawale; Ferenbach, Andrew T.; Müller, H.-Arno J.; van Aalten, Daan M. F.

    2015-01-01

    Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity. PMID:26674417

  1. Structure, function and disease relevance of Omega-class glutathione transferases.

    PubMed

    Board, Philip G; Menon, Deepthi

    2016-05-01

    The Omega-class cytosolic glutathione transferases (GSTs) have distinct structural and functional attributes that allow them to perform novel roles unrelated to the functions of other GSTs. Mammalian GSTO1-1 has been found to play a previously unappreciated role in the glutathionylation cycle that is emerging as significant mechanism regulating protein function. GSTO1-1-catalyzed glutathionylation or deglutathionylation of a key signaling protein may explain the requirement for catalytically active GSTO1-1 in LPS-stimulated pro-inflammatory signaling through the TLR4 receptor. The observation that ML175 a specific GSTO1-1 inhibitor can block LPS-stimulated inflammatory signaling has opened a new avenue for the development of novel anti-inflammatory drugs that could be useful in the treatment of toxic shock and other inflammatory disorders. The role of GSTO2-2 remains unclear. As a dehydroascorbate reductase, it could contribute to the maintenance of cellular redox balance and it is interesting to note that the GSTO2 N142D polymorphism has been associated with multiple diseases including Alzheimer's disease, Parkinson's disease, familial amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, age-related cataract and breast cancer. PMID:26993125

  2. Glutathione-S-transferase in Nereis succinea (Polychaeta) and its induction by xeno-estrogen.

    PubMed

    Ayoola, James A O; García-Alonso, Javier; Hardege, Jörg D

    2011-10-01

    The need to replace or at least to reduce the use of vertebrates in toxicity tests is a timely major concern in research and industry but to date, efforts made to minimize their use are still far from complete. Increasing demands for toxicity tests put considerable pressures upon the development of future fast and efficient test methods using invertebrates. In fact, to date, few studies provide links between biochemical and cellular effects of xeno-estrogens in aquatic invertebrates. Glutathione-S-transferase (GST) activity, as a biomarker of stress exposure, was measured in the population of clamworms (Nereis succinea) from Cardiff Bay. In addition, we examined the effect of single exposure to nonylphenol (NP) on this enzymatic activity. Field study results showed a relationship between the worm's size, reproductive status, and GST activity from the field population. In addition, we show a significant increase in the GST activity at 100 μg/L NP with sex-specific responses. The xeno-estrogens, which could affect reproduction of nereid by interfering in normal endocrinological pathways, are eliminated through GST by conjugation with glutathione. This work shows for the first time that GST activity depends on sex and stage of the clamworms and also that the xeno-estrogen NP induces its activity. This study supports the use of this species as a bioindicator of aquatic pollution and lays the foundation to causally link toxic exposure with reproductive output. PMID:20549611

  3. Development of pyrethroid-like fluorescent substrates for glutathione S-transferase

    PubMed Central

    Huang, Huazhang; Yao, Hongwei; Liu, Jun-Yan; Samra, Aman I.; Kamita, Shizuo G.; Cornel, Anthony J.; Hammock, Bruce D.

    2012-01-01

    The availability of highly sensitive substrates is critical for the development of precise and rapid assays for detecting changes in glutathione S-transferase (GST) activity that are associated with GST-mediated metabolism of insecticides. In this study, six pyrethroid-like compounds were synthesized and characterized as substrates for insect and mammalian GSTs. All of the substrates were esters composed of the same alcohol moiety, 7-hydroxy-4-methylcoumarin, and acid moieties that structurally mimic some commonly used pyrethroid insecticides including cypermethrin and cyhalothrin. CpGSTD1, a recombinant Delta class GST from the mosquito Culex pipiens, metabolized our pyrethroid-like substrates with both chemical and geometric (i.e., the cis-isomers were metabolized at 2- to 5-fold higher rates than the corresponding trans-isomers) preference. A GST preparation from mouse liver also metabolized most of our pyrethroid-like substrates with both chemical and geometric preference but at 10- to 170-fold lower rates. CpGSTD1 and mouse GSTs metabolized CDNB, a general GST substrate, at more than 200-fold higher rates than our novel pyrethroid-like substrates. There was a 10-fold difference in the specificity constant (kcat/KM ratio) of CpGSTD1 for CDNB and those of CpGSTD1 for cis-DCVC and cis-TFMCVC suggesting that cis-DCVC and cis-TFMCVC may be useful for the detection of GST-based metabolism of pyrethroids in mosquitoes. PMID:23000005

  4. Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

    PubMed Central

    Singarapu, Kiran K.; Otte, Michele M.; Tonelli, Marco; Westler, William M.; Escalante-Semerena, Jorge C.; Markley, John L.

    2015-01-01

    GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time. PMID:26513744

  5. 3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

    PubMed

    Pettigrew, N E; Brush, E J; Colman, R F

    2001-06-26

    The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects. PMID:11412109

  6. Induction of Epoxide Hydrolase, Glucuronosyl Transferase, and Sulfotransferase by Phenethyl Isothiocyanate in Male Wistar Albino Rats

    PubMed Central

    Mohd Noor, Noramaliza; Konsue, Nattaya

    2014-01-01

    Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention. PMID:24592387

  7. Methionine sulfoxide reductase regulates brain catechol-O-methyl transferase activity.

    PubMed

    Moskovitz, Jackob; Walss-Bass, Consuelo; Cruz, Dianne A; Thompson, Peter M; Bortolato, Marco

    2014-10-01

    Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined. PMID:24735585

  8. Activated Drosophila Ras1 is selectively suppressed by isoprenyl transferase inhibitors.

    PubMed Central

    Kauffmann, R C; Qian, Y; Vogt, A; Sebti, S M; Hamilton, A D; Carthew, R W

    1995-01-01

    Ras CAAX (C = cysteine, A = aliphatic amino acid, and X = any amino acid) peptidomimetic inhibitors of farnesyl protein transferase suppress Ras-dependent cell transformation by preventing farnesylation of the Ras oncoprotein. These compounds are potential anticancer agents for tumors associated with Ras mutations. The peptidomimetic FTI-254 was tested for Ras1-inhibiting activity in whole animals by injection of activated Ras1val12 Drosophila larvae. FTI-254 decreased the ability of Ras1val12 to form supernumerary R7 photoreceptor cells in the compound eye of transformed flies. In contrast, it had no effect on the related supernumerary R7 phenotypes of flies transformed with either the activated sevenless receptor tyrosine kinase, Raf kinase, or a chimeric Ras1val12 protein that is membrane associated through myristylation instead of isoprenylation. Therefore, FTI-254 acts as an isoprenylation inhibitor to selectively inhibit Ras1val12 signaling activity in a whole-animal model system. Images Fig. 2 PMID:7479910

  9. Glutathione S-transferase K1 genotype and overweight status in schizophrenia patients: A pilot study.

    PubMed

    Oniki, Kentaro; Kamihashi, Ryoko; Tomita, Tetsu; Ishioka, Masamichi; Yoshimori, Yuki; Osaki, Natsumi; Tsuchimine, Shoko; Sugawara, Norio; Kajiwara, Ayami; Morita, Kazunori; Miyata, Keishi; Otake, Koji; Nakagawa, Kazuko; Ogata, Yasuhiro; Saruwatari, Junji; Yasui-Furukori, Norio

    2016-05-30

    Elevated oxidative stress in mitochondria and mitochondrial dysfunction are associated with weight gain in schizophrenia (SCZ) patients. Glutathione S-transferase kappa 1 (GSTK1) protects cells against exogenous and endogenous oxidative stress in the mitochondria. This exploratory study investigated the possible effects of a common GSTK1 polymorphism (rs1917760, G-1308T) on the risk for overweight status among 329 SCZ patients and 305 age- and gender-matched controls and on the GSTK1 mRNA level in peripheral blood mononuclear cells among 14 SCZ patients. The GSTK1 T/T genotype was associated with having a higher BMI value among SCZ male patients, whereas this genotype tended to be associated with a lower BMI value among female patients. Conversely, these associations were not observed among the controls. The GSTK1 T/T genotype was associated with decreased GSTK1 mRNA level among SCZ patients. The GSTK1 T/T genotype may be a novel risk factor for the prediction of overweight status in SCZ male patients, although the results of this pilot study should be verified by a larger study. PMID:27010189

  10. Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis.

    PubMed

    Tu, Bin; Liu, Li; Xu, Chi; Zhai, Jixian; Li, Shengben; Lopez, Miguel A; Zhao, Yuanyuan; Yu, Yu; Ramachandran, Vanitharani; Ren, Guodong; Yu, Bin; Li, Shigui; Meyers, Blake C; Mo, Beixin; Chen, Xuemei

    2015-04-01

    3' uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2'-O-methylation on the 3' terminal ribose protects micro(mi)RNAs from 3' truncation and 3' uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3' tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA uridylyltransferase 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3' end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3' tailing could affect the activities of miRNAs in addition to leading to miRNA degradation. PMID:25928405

  11. 23S rRNA nucleotides in the peptidyl transferase center are essential for tryptophanase operon induction.

    PubMed

    Yang, Rui; Cruz-Vera, Luis R; Yanofsky, Charles

    2009-06-01

    Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNA(Pro) leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNA(Pro) by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNA(Pro) to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNA(Pro) from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression. PMID:19329641

  12. QUANTITATIVE IMAGE CYTOMETRY OF HEPATOCYTES EXPRESSING GAMMA-GLUTAMYL TRANSPEPTIDASE AND GLUTATHIONE S-TRANSFERASE IN DIETHYLNITROSAMINE-INITIATED RATS TREATED WITH PHENOBARBITAL AND/OR PHTHALATE ESTERS

    EPA Science Inventory

    Image cytometry was used to quantify the volume of liver tissue expressing two widely accepted biochemical markers of neoplasia, gammaglutamyl transpeptidase (GGT) and the placental isozyme of glutathione s-transferase (GST-P). ats were treated with hepatocarcinogen, diethylnitro...

  13. Identification of a hard surface contact-induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase, a novel fungal virulence factor.

    PubMed

    Kim, Yeon-Ki; Wang, Yuhuan; Liu, Zhi-Mei; Kolattukudy, Pappachan E

    2002-04-01

    Hard surface contact has been known to be necessary to induce infection structure (appressorium) formation in many phytopathogenic fungi. However, the molecular basis of this requirement is unknown. We have used a differential display approach to clone some of the genes induced in the conidia by hard surface contact. We report that one of the genes induced by hard-surface contact of the conidia of Colletotrichum gloeosporioides, chip6, encodes a protein with homology to sterol glycosyl transferases. chip6 expressed in E. coli catalyses glucosyl transfer from UDP-glucose to cholesterol. Disruption of chip6 causes a marked decrease in the transferase activity and a drastic reduction in virulence on its natural host, avocado fruits, although the mutant is capable of normal growth and appressorium formation. The requirement for sterol glycosyl transferase for pathogenicity suggests a novel biological function for this transferase. PMID:12000454

  14. Neuroantibodies (NAB) in African-American Children: Associations with Gender, Glutathione-S-Transferase (GST)Pi Polymorphisms (SNP) and Heavy Metals

    EPA Science Inventory

    CONTACT (NAME ONLY): Hassan El-Fawal Abstract Details PRESENTATION TYPE: Platform or Poster CURRENT CATEGORY: Neurodegenerative Disease | Biomarkers | Neurotoxicity, Metals KEYWORDS: Autoantibodies, Glutathione-S-Transferase, DATE/TIME LAST MODIFIED: DATE/TIME SUBMITTED: Abs...

  15. SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation.

    PubMed

    Grishina, Inna; Debus, Katherina; García-Limones, Carmen; Schneider, Constanze; Shresta, Amit; García, Carlos; Calzado, Marco A; Schmitz, M Lienhard

    2012-12-01

    Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response. PMID:23044042

  16. A shotgun lipidomics study of a putative lysophosphatidic acid acyl transferase (PlsC) in Sinorhizobium meliloti.

    PubMed

    Basconcillo, Libia Saborido; Zaheer, Rahat; Finan, Turlough M; McCarry, Brian E

    2009-09-15

    A shotgun lipidomics approach was used to study the knockout mutant of a putative lysophosphatidic acyl acid transferase (PlsC) in order to delineate the function of this enzyme in Sinorhizobium meliloti. In plsC knockout mutant lipids that contained 16:0 and 16:1 fatty acids and their biosynthetically related cyclopropane fatty acid (cis-9,10-methylene hexadecanoic acid) decreased up to 93%. Tandem mass spectrometry experiments in the presence of added Li(+) showed that the putative PlsC (SMc00714) functioned as a lysophosphatidic acid acyl transferase specific for the transfer of C16 fatty acids to the sn-2 position of lipids. The levels of lipids containing C18 fatty acids were unaffected in plsC mutant, suggesting the presence of one or more fatty acyl transferases in the genome of S. meliloti with selectivity towards C18 fatty acids. Two non-phosphorus containing lipid classes, sulfoquinovosyldiacylglycerol and 1,2-diacylglyceryl-trimethylhomoserine lipids, showed similar decreases in C16 fatty acid content as phospholipids in plsC knockout mutant; these non-phosphorus containing lipids share a common biosynthetic origin with phospholipids, most likely involving phosphatidic acid. Ornithine lipids containing C16 fatty acids also showed decreased levels in PlsC knockout mutant, suggesting that PlsC is also involved in their biosynthesis. PMID:19525157

  17. Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice

    SciTech Connect

    Conklin, Daniel J.; Haberzettl, Petra; Jagatheesan, Ganapathy; Baba, Shahid; Merchant, Michael L.; Prough, Russell A.; Williams, Jessica D.; Prabhu, Sumanth D.; Bhatnagar, Aruni

    2015-06-01

    High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null than WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. - Graphical abstract: Cyclophosphamide (CY) treatment results in P450-mediated metabolic formation of phosphoramide mustard and acrolein (3-propenal). Acrolein is either metabolized and

  18. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed Central

    Fotouhi-Ardakani, N; Batist, G

    1999-01-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  19. Identification of glutathione S-transferase genes responding to pathogen infestation in Populus tomentosa.

    PubMed

    Liao, Weihua; Ji, Lexiang; Wang, Jia; Chen, Zhong; Ye, Meixia; Ma, Huandi; An, Xinmin

    2014-09-01

    Stem blister canker, caused by Botryosphaeria dothidea, is becoming the most serious disease of poplar in China. The molecular basis of the poplar in response to stem blister canker is not well understood. To reveal the global transcriptional changes of poplar to infection by B. dothidea, Solexa paired-end sequencing of complementary DNAs (cDNAs) from control (NB) and pathogen-treated samples (WB) was performed, resulting in a total of 339,283 transcripts and 183,881 unigenes. A total of 206,586 transcripts were differentially expressed in response to pathogen stress (false discovery rate ≤0.05 and an absolute value of log2Ratio (NB/WB) ≥1). In enrichment analysis, energy metabolism and redox reaction-related macromolecules were accumulated significantly in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses, indicating components of dynamic defense against the fungus. A total of 852 transcripts (575 upregulated and 277 downregulated transcripts) potentially involved in plant-pathogen interaction were also differentially regulated, including genes encoding proteins linked to signal transduction (putative leucine-rich repeat (LRR) protein kinases and calcium-binding proteins), defense (pathogenesis-related protein 1), and cofactors (jasmonate-ZIM-domain-containing proteins and heat shock proteins). Moreover, transcripts encoding glutathione S-transferase (GST) were accumulated to high levels, revealing key genes and proteins potentially related to pathogen resistance. Poplar RNA sequence data were validated by quantitative real-time PCR (RT-qPCR), which revealed a highly reliability of the transcriptomic profiling data. PMID:24870810

  20. Activities of the peptidyl transferase center of ribosomes lacking protein L27

    PubMed Central

    Maracci, Cristina; Wohlgemuth, Ingo; Rodnina, Marina V.

    2015-01-01

    The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome. PMID:26475831

  1. Increased transcription of Glutathione S-transferases in acaricide exposed scabies mites

    PubMed Central

    2010-01-01

    Background Recent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches. Results Increased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and P-glycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment. Conclusions These findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite. PMID:20482766

  2. Characterization and Functional Analysis of Four Glutathione S-Transferases from the Migratory Locust, Locusta migratoria

    PubMed Central

    Qin, Guohua; Jia, Miao; Liu, Ting; Zhang, Xueyao; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2013-01-01

    Glutathione S-transferases (GSTs) play an important role in detoxification of xenobiotics in both prokaryotic and eukaryotic cells. In this study, four GSTs (LmGSTd1, LmGSTs5, LmGSTt1, and LmGSTu1) representing different classes were identified from the migratory locust, Locusta migratoria. These four proteins were heterologously expressed in Escherichia coli as soluble fusion proteins, purified by Ni2+-nitrilotriacetic acid agarose column and biochemically characterized. LmGSTd1, LmGSTs5, and LmGSTu1 showed high activities with 1-chloro-2, 4-dinitrobenzene (CDNB), detectable activity with p-nitro-benzyl chloride (p-NBC) and 1, 2-dichloro-4-nitrobenzene (DCNB), whereas LmGSTt1 showed high activity with p-NBC and detectable activity with CDNB. The optimal pH of the locust GSTs ranged between 7.0 to 9.0. Ethacrynic acid and reactive blue effectively inhibited all four GSTs. LmGSTs5 was most sensitive to heavy metals (Cu2+ and Cd2+). The maximum expression of the four GSTs was observed in Malpighian tubules and fat bodies as evaluated by western blot. The nymph mortalities after carbaryl treatment increased by 28 and 12% after LmGSTs5 and LmGSTu1 were silenced, respectively. The nymph mortalities after malathion and chlorpyrifos treatments increased by 26 and 18% after LmGSTs5 and LmGSTu1 were silenced, respectively. These results suggest that sigma GSTs in L. migratoria play a significant role in carbaryl detoxification, whereas some of other GSTs may also involve in the detoxification of carbaryl and chlorpyrifos. PMID:23505503

  3. Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

    PubMed Central

    Ralat, Luis A.; Misquitta, Stephanie A.; Manevich, Yefim; Fisher, Aron B.; Colman, Roberta F.

    2016-01-01

    Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the Mw of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The Mw of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the Km value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41–85, 115–124, 131–163, and 164–197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer. PMID:18358825

  4. Genetic polymorphism for glutathione-S-transferase mu in asbestos cement workers.

    PubMed Central

    Jakobsson, K; Rannug, A; Alexandrie, A K; Rylander, L; Albin, M; Hagmar, L

    1994-01-01

    OBJECTIVE--To investigate whether a lack of glutathione-S-transferase mu (GSTM1) activity was related to an increased risk for adverse outcome after asbestos exposure. METHODS--A study was made of 78 male former asbestos cement workers, with retrospective cohort data on exposure, radiographical findings, and lung function. Venous blood samples were obtained for the analysis of GSTM1 polymorphism by the polymerase chain reaction technique. Chest x ray films were classified according to the International Labour Organisation (ILO) 1980 classification. Vital capacity (VC) and forced expiratory volume during 1 s (FEV1) were determined. Individual estimates of asbestos exposure were calculated, and expressed as duration of exposure, average exposure intensity, and cumulative dose. Data on smoking were obtained from interviews. RESULTS--The lung function in the study group was reduced, compared with reference equations. 23% of the workers had small opacities > or = 1/0, 29% circumscribed pleural thickenings, 14% diffuse thickenings, and 12% obliterated costophrenic angles. 54% of the workers were GSTM1 deficient. They were comparable with the other workers in age, follow up time (median 30 years), and duration of exposure (median 18 years), but had a slightly higher cumulated dose (median 18 v 10 fibre-years) than the others. Neither in radiographical changes nor lung function variables were there any differences between the different GSTM1 groups. The findings were similar when smoking habits and estimated asbestos exposure were taken into account. CONCLUSIONS--We could not show that lack of GSTM1 activity was related to an increased risk for radiographical or lung function changes in a group of asbestos cement workers, followed up for a long period after the end of exposure. PMID:7849864

  5. Urinary π-glutathione S-transferase Predicts Advanced Acute Kidney Injury Following Cardiovascular Surgery.

    PubMed

    Shu, Kai-Hsiang; Wang, Chih-Hsien; Wu, Che-Hsiung; Huang, Tao-Min; Wu, Pei-Chen; Lai, Chien-Heng; Tseng, Li-Jung; Tsai, Pi-Ru; Connolly, Rory; Wu, Vin-Cent

    2016-01-01

    Urinary biomarkers augment the diagnosis of acute kidney injury (AKI), with AKI after cardiovascular surgeries being a prototype of prognosis scenario. Glutathione S-transferases (GST) were evaluated as biomarkers of AKI. Urine samples were collected in 141 cardiovascular surgical patients and analyzed for urinary alpha-(α-) and pi-(π-) GSTs. The outcomes of advanced AKI (KDIGO stage 2, 3) and all-cause in-patient mortality, as composite outcome, were recorded. Areas under the receiver operator characteristic (ROC) curves and multivariate generalized additive model (GAM) were applied to predict outcomes. Thirty-eight (26.9%) patients had AKI, while 12 (8.5%) were with advanced AKI. Urinary π-GST differentiated patients with/without advanced AKI or composite outcome after surgery (p < 0.05 by generalized estimating equation). Urinary π-GST predicted advanced AKI at 3 hrs post-surgery (p = 0.033) and composite outcome (p = 0.009), while the corresponding ROC curve had AUC of 0.784 and 0.783. Using GAM, the cutoff value of 14.7 μg/L for π-GST showed the best performance to predict composite outcome. The addition of π-GST to the SOFA score improved risk stratification (total net reclassification index = 0.47). Thus, urinary π-GST levels predict advanced AKI or hospital mortality after cardiovascular surgery and improve in SOFA outcome assessment specific to AKI. PMID:27527370

  6. Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

    PubMed Central

    Jemth, P; Stenberg, G; Chaga, G; Mannervik, B

    1996-01-01

    Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10). PMID:8645195

  7. Busulfan conjugation by glutathione S-transferases alpha, mu, and pi.

    PubMed

    Czerwinski, M; Gibbs, J P; Slattery, J T

    1996-09-01

    Busulfan is eliminated by glutathione S-transferase (GST)-catalyzed conjugation with glutathione (GSH). We have characterized the busulfan-conjugating activity of purified human liver GSTA1-1, GSTA1-2, GSTA2-2, GSTM1-1, and placental GSTP1-1. Isoforms were purified from cytosol by GSH-affinity chromatography and chromatofocusing. In addition, the busulfan-conjugating activity of cDNA-expressed GTH1 and GTH2, corresponding to GSTA1-1 and GSTA2-2, were characterized. The major product of busulfan conjugation, a thiophenium ion (THT+), was assayed by GC/MS after conversion to tetrahydrothiophene (THT). THT+ formation rate increased linearly with busulfan concentration up to its solubility limit for all GST isoforms. Because Vmax and KM could not be determined separately, the slope of the velocity vs. substrate concentration plot, Vmax/KM was used to compare isoform activities. Vmax/KM for GSTA1-1 was 7.95 microliters/min/mg protein, the highest busulfan-conjugating activity of all human liver and placenta isoforms evaluated. GSTM1-1 and GSTP1-1, respectively, had 46% and 18% of the activity of GSTA1-1. Since the polymorphic mu-class GST catalyzed busulfan conjugation, we examined busulfan clearance in 50 patients undergoing high-dose busulfan before bone marrow transplantation. Busulfan clearance was normally distributed, suggesting that GSTM1-1 does not contribute significantly to the elimination of busulfan from the body. We conclude that GSTA1-1 is the major isoform catalyzing busulfan conjugation, whereas GSTM1-1 and GSTP1-1 may be important in the protection of specific cells. PMID:8886613

  8. Comparison of human liver and small intestinal glutathione S-transferase-catalyzed busulfan conjugation in vitro.

    PubMed

    Gibbs, J P; Yang, J S; Slattery, J T

    1998-01-01

    The apparent oral clearance of busulfan has been observed to vary as much as 10-fold in the population of children and adults receiving high-dose busulfan. The only identified elimination pathway for busulfan involves glutathione conjugation. The reaction is predominantly catalyzed by glutathione S-transferase (GST) A1-1, which is present in both liver and intestine. The purpose of this study was to compare busulfan Vmax/Km in cytosol prepared from adult human liver and small intestine. Tetrahydrothiophenium ion formation rate per milligram of cytosolic protein was constant along the length (assessed in 30-cm segments) of three individual small intestines. A 30-cm-long intestinal segment 90-180 cm from the pylorus was chosen to be representative of intestinal cytosolic busulfan conjugating activity. Busulfan Vmax/Km (mean +/- SD) in cytosol prepared from 23 livers and 12 small intestines was 0.166 +/- 0.066 and 0.176 +/- 0.085 microl/min/mg cytosolic protein, respectively, in incubations with 5 microM busulfan, 1 mM glutathione, and 2 mg of cytosolic protein. The relative content of GSTalpha (A1-1, A1-2, and A2-2) was compared for human liver and intestinal cytosol using Western blot. The levels of GSTalpha in liver and intestinal cytosol were 1.12 +/- 0.56 and 1.36 +/- 0.32 integrated optimal density units/5 microg cytosolic protein, respectively. Busulfan conjugation in vitro was comparable per milligram of cytosolic protein in liver and intestinal cytosol. PMID:9443852

  9. Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1

    PubMed Central

    YANG, LIANG; LIU, REN; MA, HONG-BIN; YING, MING-ZHEN; WANG, YA-JIE

    2015-01-01

    The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 (GSTP1) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G2/M phase arrest in the GSTP1-expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1-expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G2/M phase arrest. PMID:26622693

  10. The Sigma Class Glutathione Transferase from the Liver Fluke Fasciola hepatica

    PubMed Central

    LaCourse, E. James; Perally, Samirah; Morphew, Russell M.; Moxon, Joseph V.; Prescott, Mark; Dowling, David J.; O'Neill, Sandra M.; Kipar, Anja; Hetzel, Udo; Hoey, Elizabeth; Zafra, Rafael; Buffoni, Leandro; Pérez Arévalo, José; Brophy, Peter M.

    2012-01-01

    Background Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. Methodology/Principal Findings In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. Conclusions/Significance We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity. PMID:22666515

  11. The Carnitine Palmitoyl Transferase (CPT) System and Possible Relevance for Neuropsychiatric and Neurological Conditions.

    PubMed

    Virmani, Ashraf; Pinto, Luigi; Bauermann, Otto; Zerelli, Saf; Diedenhofen, Andreas; Binienda, Zbigniew K; Ali, Syed F; van der Leij, Feike R

    2015-10-01

    The carnitine palmitoyl transferase (CPT) system is a multiprotein complex with catalytic activity localized within a core represented by CPT1 and CPT2 in the outer and inner membrane of the mitochondria, respectively. Two proteins, the acyl-CoA synthase and a translocase also form part of this system. This system is crucial for the mitochondrial beta-oxidation of long-chain fatty acids. CPT1 has two well-known isoforms, CPT1a and CPT1b. CPT1a is the hepatic isoform and CPT1b is typically muscular; both are normally utilized by the organism for metabolic processes throughout the body. There is a strong evidence for their involvement in various disease states, e.g., metabolic syndrome, cardiovascular diseases, and in diabetes mellitus type 2. Recently, a new, third isoform of CPT was described, CPT1c. This is a neuronal isoform and is prevalently localized in brain regions such as hypothalamus, amygdala, and hippocampus. These brain regions play an important role in control of food intake and neuropsychiatric and neurological diseases. CPT activity has been implicated in several neurological and social diseases mainly related to the alteration of insulin equilibrium in the brain. These pathologies include Parkinson's disease, Alzheimer's disease, and schizophrenia. Evolution of both Parkinson's disease and Alzheimer's disease is in some way linked to brain insulin and related metabolic dysfunctions with putative links also with the diabetes type 2. Studies show that in the CNS, CPT1c affects ceramide levels, endocannabionoids, and oxidative processes and may play an important role in various brain functions such as learning. PMID:26041663

  12. Glutathione-supported arsenate reduction coupled to arsenolysis catalyzed by ornithine carbamoyl transferase

    SciTech Connect

    Nemeti, Balazs; Gregus, Zoltan

    2009-09-01

    Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.

  13. The still mysterious roles of cysteine-containing glutathione transferases in plants

    PubMed Central

    Lallement, Pierre-Alexandre; Brouwer, Bastiaan; Keech, Olivier; Hecker, Arnaud; Rouhier, Nicolas

    2014-01-01

    Glutathione transferases (GSTs) represent a widespread multigenic enzyme family able to modify a broad range of molecules. These notably include secondary metabolites and exogenous substrates often referred to as xenobiotics, usually for their detoxification, subsequent transport or export. To achieve this, these enzymes can bind non-substrate ligands (ligandin function) and/or catalyze the conjugation of glutathione onto the targeted molecules, the latter activity being exhibited by GSTs having a serine or a tyrosine as catalytic residues. Besides, other GST members possess a catalytic cysteine residue, a substitution that radically changes enzyme properties. Instead of promoting GSH-conjugation reactions, cysteine-containing GSTs (Cys-GSTs) are able to perform deglutathionylation reactions similarly to glutaredoxins but the targets are usually different since glutaredoxin substrates are mostly oxidized proteins and Cys-GST substrates are metabolites. The Cys-GSTs are found in most organisms and form several classes. While Beta and Omega GSTs and chloride intracellular channel proteins (CLICs) are not found in plants, these organisms possess microsomal ProstaGlandin E-Synthase type 2, glutathionyl hydroquinone reductases, Lambda, Iota and Hemerythrin GSTs and dehydroascorbate reductases (DHARs); the four last classes being restricted to the green lineage. In plants, whereas the role of DHARs is clearly associated to the reduction of dehydroascorbate to ascorbate, the physiological roles of other Cys-GSTs remain largely unknown. In this context, a genomic and phylogenetic analysis of Cys-GSTs in photosynthetic organisms provides an updated classification that is discussed in the light of the recent literature about the functional and structural properties of Cys-GSTs. Considering the antioxidant potencies of phenolic compounds and more generally of secondary metabolites, the connection of GSTs with secondary metabolism may be interesting from a pharmacological

  14. The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center.

    PubMed

    Englander, Michael T; Avins, Joshua L; Fleisher, Rachel C; Liu, Bo; Effraim, Philip R; Wang, Jiangning; Schulten, Klaus; Leyh, Thomas S; Gonzalez, Ruben L; Cornish, Virginia W

    2015-05-12

    The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells. PMID:25918365

  15. The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

    PubMed Central

    Englander, Michael T.; Avins, Joshua L.; Fleisher, Rachel C.; Liu, Bo; Effraim, Philip R.; Wang, Jiangning; Schulten, Klaus; Leyh, Thomas S.; Gonzalez, Ruben L.; Cornish, Virginia W.

    2015-01-01

    The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells. PMID:25918365

  16. The poplar Phi class glutathione transferase: expression, activity and structure of GSTF1

    PubMed Central

    Pégeot, Henri; Koh, Cha San; Petre, Benjamin; Mathiot, Sandrine; Duplessis, Sébastien; Hecker, Arnaud; Didierjean, Claude; Rouhier, Nicolas

    2014-01-01

    Glutathione transferases (GSTs) constitute a superfamily of enzymes with essential roles in cellular detoxification and secondary metabolism in plants as in other organisms. Several plant GSTs, including those of the Phi class (GSTFs), require a conserved catalytic serine residue to perform glutathione (GSH)-conjugation reactions. Genomic analyses revealed that terrestrial plants have around ten GSTFs, eight in the Populus trichocarpa genome, but their physiological functions and substrates are mostly unknown. Transcript expression analyses showed a predominant expression of all genes both in reproductive (female flowers, fruits, floral buds) and vegetative organs (leaves, petioles). Here, we show that the recombinant poplar GSTF1 (PttGSTF1) possesses peroxidase activity toward cumene hydroperoxide and GSH-conjugation activity toward model substrates such as 2,4-dinitrochlorobenzene, benzyl and phenetyl isothiocyanate, 4-nitrophenyl butyrate and 4-hydroxy-2-nonenal but interestingly not on previously identified GSTF-class substrates. In accordance with analytical gel filtration data, crystal structure of PttGSTF1 showed a canonical dimeric organization with bound GSH or 2-(N-morpholino)ethanesulfonic acid molecules. The structure of these protein-substrate complexes allowed delineating the residues contributing to both the G and H sites that form the active site cavity. In sum, the presence of GSTF1 transcripts and proteins in most poplar organs especially those rich in secondary metabolites such as flowers and fruits, together with its GSH-conjugation activity and its documented stress-responsive expression suggest that its function is associated with the catalytic transformation of metabolites and/or peroxide removal rather than with ligandin properties as previously reported for other GSTFs. PMID:25566286

  17. Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

    PubMed Central

    Alfenito, M R; Souer, E; Goodman, C D; Buell, R; Mol, J; Koes, R; Walbot, V

    1998-01-01

    Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1. PMID:9668133

  18. Glutathione-S-transferase profiles in the emerald ash borer, Agrilus planipennis.

    PubMed

    Rajarapu, Swapna Priya; Mittapalli, Omprakash

    2013-05-01

    The emerald ash borer, Agrilus planipennis Fairmaire is a recently discovered invasive insect pest of ash, Fraxinus spp. in North America. Glutathione-S-transferases (GST) are a multifunctional superfamily of enzymes which function in conjugating toxic compounds to less toxic and excretable forms. In this study, we report the molecular characterization and expression patterns of different classes of GST genes in different tissues and developmental stages plus their specific activity. Multiple sequence alignment of all six A. planipennis GSTs (ApGST-E1, ApGST-E2, ApGST-E3, ApGST-O1, ApGST-S1 and ApGST-μ1) revealed conserved features of insect GSTs and a phylogenetic analysis grouped the GSTs within the epsilon, sigma, omega and microsomal classes of GSTs. Real time quantitative PCR was used to study field collected samples. In larval tissues high mRNA levels for ApGST-E1, ApGST-E3 and ApGST-O1 were obtained in the midgut and Malpighian tubules. On the other hand, ApGST-E2 and ApGST-S1 showed high mRNA levels in fat body and ApGST-μ1 showed constitutive levels in all the tissues assayed. During development, mRNA levels for ApGST-E2 were observed to be the highest in feeding instars, ApGST-S1 in prepupal instars; while the others showed constitutive patterns in all the developmental stages examined. At the enzyme level, total GST activity was similar in all the tissues and developmental stages assayed. Results obtained suggest that A. planipennis is potentially primed with GST-driven detoxification to metabolize ash allelochemicals. To our knowledge this study represents the first report of GSTs in A. planipennis and also in the family of wood boring beetles. PMID:23499941

  19. Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon

    PubMed Central

    Espinoza, Herbert M.; Shireman, Laura M.; McClain, Valerie; Atkins, William; Gallagher, Evan P.

    2013-01-01

    The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720 bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 727 and 681 bp, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655 Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity towards 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were KM = 0.16 ± 0.06 mM and Vmax = 0.5 ± 0.1 μmol min−1 mg−1 for OlfGST pi, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (KM = 0.022 ± 0.008 mM and Vmax = 0.47 ± 0.05 μmol min−1 mg−1). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox statusand signal transduction. PMID:23261526

  20. Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

    PubMed

    Espinoza, Herbert M; Shireman, Laura M; McClain, Valerie; Atkins, William; Gallagher, Evan P

    2013-03-15

    The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 627 and 681nt, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity toward 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were K(M)=0.16 ± 0.06mM and V(max)=0.5 ± 0.1μmolmin⁻¹mg⁻¹, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (K(M)=0.022 ± 0.008 mM and V(max)=0.47 ± 0.05μmolmin⁻¹mg⁻¹). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox status and signal transduction. PMID:23261526

  1. The stereochemical course of 4-hydroxy-2-nonenal metabolism by glutathione S-transferases.

    PubMed

    Balogh, Larissa M; Roberts, Arthur G; Shireman, Laura M; Greene, Robert J; Atkins, William M

    2008-06-13

    4-Hydroxy-2-nonenal (HNE) is a toxic aldehyde generated during lipid peroxidation and has been implicated in a variety of pathological states associated with oxidative stress. Glutathione S-transferase (GST) A4-4 is recognized as one of the predominant enzymes responsible for the metabolism of HNE. However, substrate and product stereoselectivity remain to be fully explored. The results from a product formation assay indicate that hGSTA4-4 exhibits a modest preference for the biotransformation of S-HNE in the presence of both enantiomers. Liquid chromatography mass spectrometry analyses using the racemic and enantioisomeric HNE substrates explicitly demonstrate that hGSTA4-4 conjugates glutathione to both HNE enantiomers in a completely stereoselective manner that is not maintained in the spontaneous reaction. Compared with other hGST isoforms, hGSTA4-4 shows the highest degree of stereoselectivity. NMR experiments in combination with simulated annealing structure determinations enabled the determination of stereochemical configurations for the GSHNE diastereomers and are consistent with an hGSTA4-4-catalyzed nucleophilic attack that produces only the S-configuration at the site of conjugation, regardless of substrate chirality. In total these results indicate that hGSTA4-4 exhibits an intriguing combination of low substrate stereoselectivity with strict product stereoselectivity. This behavior allows for the detoxification of both HNE enantiomers while generating only a select set of GSHNE diastereomers with potential stereochemical implications concerning their effects and fates in biological tissues. PMID:18424441

  2. Cloning, expression and identification of two glutathione S-transferase isoenzymes from Perna viridis.

    PubMed

    Li, Zhenzhen; Chen, Rong; Zuo, Zhenghong; Mo, Zhengping; Yu, Ang

    2013-08-01

    Glutathione S-transferases (GSTs; EC 2.5.1.18) are phase II enzymes involved in major detoxification reactions of xenobiotic in many organisms. In the present study, two classes of GSTs (PvGST1 and PvGST2) were cloned from P. viridis by rapid amplification of cDNA ends method. Sequence alignments and phylogenetic analysis together supported that PvGST1 and PvGST2 belonged to the pi and omega classes, respectively. The PvGST1 cDNA was 1214 nucleotides (nt) in length and contained a 618 nt open reading frame (ORF) encoding 206 amino acid residues, and had 46 nt of 5'-untranslated region (UTR) and a 3' UTR of 550 nt including a tailing signal (AATAAA) and a poly (A) tail. The molecular mass of the predicted PvGST1 was 23.815kDa, with the calculated isoelectric point being 5.39. PvGST2 was 1093bp, consisting of a 5' UTR of 13bp, a 3' UTR of 246bp and an ORF of 834bp. The deduced protein was composed of 278 amino acids, with an estimated molecular mass of 32.476kDa and isoelectric point of 8.88. Tissue distribution analysis of the PvGST1 and PvGST2 mRNA revealed that the GST expression level was higher in digestive gland and gonad, while lower in gill and mantle in both genders. Molecular modeling analysis of two GSTs implicated their various functions account for their different enzymatic features. PMID:23711756

  3. Nuclear morphometry and glutathione S-transferase pi expression in breast cancer.

    PubMed

    Huang, J; Bay, B H; Tan, P H

    2000-01-01

    Glutathione S-transferase pi (GST-pi) is a phase II detoxification enzyme whose expression is increased in estrogen receptor (ER)-poor breast cancers and in breast cancers resistant to certain chemotherapeutic agents. The aim of this study was to investigate the immunohistochemical expression of GST-pi in invasive breast carcinoma and to correlate the findings with those of nuclear morphometry. Formalin-fixed paraffin-embedded tissue specimens obtained from 21 invasive breast cancers and 16 adjacent (benign) tissues were immunohistochemically stained using polyclonal anti-human GST-pi antibody. There was positive (defined as >10% immunoreactive tumor cells) but variable expression of GST-pi in 10 (48%) cases. Nuclear morphometry in these 10 tumors revealed immunoreactive malignant cells to be larger (mean area 41.7+/-1.0 microm2) and more rounded in form when compared with non-staining cancer cells (mean area 28.7+/-0.7 microm2). It was also observed that GST-pi immunonegative tumor cells in GST-pi expressing tumors had different morphologies from malignant cells in the remaining 11 (52%) cancers that were regarded as GST-pi negative. Increased GST-pi expression determined by the percentage of positively staining tumor cells, was found to be significantly correlated with increased variability in nuclear area and perimeter (Spearman's rho=0.821, p=0.044 for both) in the subset of node-positive tumors. Our findings suggest that there exists two sub-populations of cancer cells with distinct nuclear morphologies in GST-pi positive tumors; factors other than GST-pi expression are likely to have a phenotypic effect on breast cancer cells; and there may be a special significance of this enzyme in axillary node-positive breast tumors. PMID:10767377

  4. Inhibitory effects of plant polyphenols on rat liver glutathione S-transferases.

    PubMed

    Zhang, K; Das, N P

    1994-06-01

    Several novel naturally occurring flavonoids and other polyphenols exerted varying degrees of concentration-dependent inhibition on uncharacterized rat liver glutathione S-transferase (EC 2.5.1.18, GST) isoforms. The order of inhibitory potencies of the five most potent polyphenols was tannic acid > 2-hydroxyl chalcone > butein > morin > quercetin, and their IC50 values were 1.044, 6.758, 9.033, 13.710 and 18.732 microM, respectively. Their inhibitions were reversible, as indicated by dialysis experiments. The optimum pH for the inhibitions by four of the compounds (tannic acid, butein, 2-hydroxyl chalcone and morin) was in the range of pH 6.0 to 6.5, but for quercetin the optimum pH was 8.0. These potent inhibitors possess one or more of the following chemical structural features: (a) polyhydroxylation substitutions, (b) absence of a sugar moiety, (c) for the chalcones, the presence of an open C-ring and hydroxylation at either the C-2 or C-3 position, (d) for the flavonoids, the attachment of the B-ring to C-2, and (e) a double bond between C-2 and C-3. Butein exhibited a non-competitive inhibition toward both glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Interestingly, tannic acid showed a non-competitive inhibition toward CDNB but a competitive inhibition toward GSH. The inhibitory potency of tannic acid on rat liver GSTs was concentration and substrate dependent. Using CDNB, p-nitrobenzyl chloride, 4-nitropyridine-N-oxide, and ethacrynic acid as substrates, the IC50 values for tannic acid were 1.044, 11.151, 20.206, and 57.664 microM, respectively. PMID:8010991

  5. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin.

    PubMed

    Eudes, Aymerick; Pereira, Jose H; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E K; Lee, Taek Soon; Adams, Paul D; Keasling, Jay D; Loqué, Dominique

    2016-03-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  6. The poplar Phi class glutathione transferase: expression, activity and structure of GSTF1.

    PubMed

    Pégeot, Henri; Koh, Cha San; Petre, Benjamin; Mathiot, Sandrine; Duplessis, Sébastien; Hecker, Arnaud; Didierjean, Claude; Rouhier, Nicolas

    2014-01-01

    Glutathione transferases (GSTs) constitute a superfamily of enzymes with essential roles in cellular detoxification and secondary metabolism in plants as in other organisms. Several plant GSTs, including those of the Phi class (GSTFs), require a conserved catalytic serine residue to perform glutathione (GSH)-conjugation reactions. Genomic analyses revealed that terrestrial plants have around ten GSTFs, eight in the Populus trichocarpa genome, but their physiological functions and substrates are mostly unknown. Transcript expression analyses showed a predominant expression of all genes both in reproductive (female flowers, fruits, floral buds) and vegetative organs (leaves, petioles). Here, we show that the recombinant poplar GSTF1 (PttGSTF1) possesses peroxidase activity toward cumene hydroperoxide and GSH-conjugation activity toward model substrates such as 2,4-dinitrochlorobenzene, benzyl and phenetyl isothiocyanate, 4-nitrophenyl butyrate and 4-hydroxy-2-nonenal but interestingly not on previously identified GSTF-class substrates. In accordance with analytical gel filtration data, crystal structure of PttGSTF1 showed a canonical dimeric organization with bound GSH or 2-(N-morpholino)ethanesulfonic acid molecules. The structure of these protein-substrate complexes allowed delineating the residues contributing to both the G and H sites that form the active site cavity. In sum, the presence of GSTF1 transcripts and proteins in most poplar organs especially those rich in secondary metabolites such as flowers and fruits, together with its GSH-conjugation activity and its documented stress-responsive expression suggest that its function is associated with the catalytic transformation of metabolites and/or peroxide removal rather than with ligandin properties as previously reported for other GSTFs. PMID:25566286

  7. Genetic polymorphism in three glutathione s-transferase genes and breast cancer risk

    SciTech Connect

    Woldegiorgis, S.; Ahmed, R.C.; Zhen, Y.; Erdmann, C.A.; Russell, M.L.; Goth-Goldstein, R.

    2002-04-01

    The role of the glutathione S-transferase (GST) enzyme family is to detoxify environmental toxins and carcinogens and to protect organisms from their adverse effects, including cancer. The genes GSTM1, GSTP1, and GSTT1 code for three GSTs involved in the detoxification of carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and benzene. In humans, GSTM1 is deleted in about 50% of the population, GSTT1 is absent in about 20%, whereas the GSTP1 gene has a single base polymorphism resulting in an enzyme with reduced activity. Epidemiological studies indicate that GST polymorphisms increase the level of carcinogen-induced DNA damage and several studies have found a correlation of polymorphisms in one of the GST genes and an increased risk for certain cancers. We examined the role of polymorphisms in genes coding for these three GST enzymes in breast cancer. A breast tissue collection consisting of specimens of breast cancer patients and non-cancer controls was analyzed by polymerase chain reaction (PCR) for the presence or absence of the GSTM1 and GSTT1 genes and for GSTP1 single base polymorphism by PCR/RFLP. We found that GSTM1 and GSTT1 deletions occurred more frequently in cases than in controls, and GSTP1 polymorphism was more frequent in controls. The effective detoxifier (putative low-risk) genotype (defined as presence of both GSTM1 and GSTT1 genes and GSTP1 wild type) was less frequent in cases than controls (16% vs. 23%, respectively). The poor detoxifier (putative high-risk) genotype was more frequent in cases than controls. However, the sample size of this study was too small to provide conclusive results.

  8. Structural analysis of an epsilon-class glutathione transferase from housefly, Musca domestica.

    PubMed

    Nakamura, Chihiro; Yajima, Shunsuke; Miyamoto, Toru; Sue, Masayuki

    2013-01-25

    Glutathione transferases (GSTs) play an important role in the detoxification of insecticides, and as such, they are a key contributor to enhanced resistance to insecticides. In the housefly (Musca domestica), two epsilon-class GSTs (MdGST6A and MdGST6B) that share high sequence homology have been identified, which are believed to be involved in resistance against insecticides. The structural determinants controlling the substrate specificity and enzyme activity of MdGST6s are unknown. The aim of this study was to crystallize and perform structural analysis of the GST isozyme, MdGST6B. The crystal structure of MdGST6B complexed with reduced glutathione (GSH) was determined at a resolution of 1.8 Å. MdGST6B was found to have a typical GST folding comprised of N-terminal and C-terminal domains. Arg113 and Phe121 on helix 4 were shown to protrude into the substrate binding pocket, and as a result, the entrance of the substrate binding pocket was narrower compared to delta- and epsilon-class GSTs from Africa malaria vector Anopheles gambiae, agGSTd1-6 and agGSTe2, respectively. This substrate pocket narrowing is partly due to the presence of a π-helix in the middle of helix 4. Among the six residues that donate hydrogen bonds to GSH, only Arg113 was located in the C-terminal domain. Ala substitution of Arg113 did not have a significant effect on enzyme activity, suggesting that the Arg113 hydrogen bond does not play a crucial role in catalysis. On the other hand, mutation at Phe108, located just below Arg113 in the binding pocket, reduced the affinity and catalytic activity to both GSH and the electrophilic co-substrate, 1-chloro-2,4-dinitrobenzene. PMID:23268341

  9. Effects of Local Heart Irradiation in a Glutathione S-Transferase Alpha 4-Null Mouse Model

    PubMed Central

    Boerma, Marjan; Singh, Preeti; Sridharan, Vijayalakshmi; Tripathi, Preeti; Sharma, Sunil; Singh, Sharda P.

    2015-01-01

    Glutathione S-transferase alpha 4 (GSTA4-4) is one of the enzymes responsible for the removal of 4-hydroxynonenal (4-HNE), an electrophilic product of lipid peroxidation in cellular membranes during oxidative stress. 4-HNE is a direct activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor with many target genes encoding antioxidant and anti-electrophile enzymes. We have previously shown that Gsta4-null mice on a 129/Sv background exhibited increased activity of Nrf2 in the heart. Here we examined the sensitivity of this Gsta4-null mouse model towards cardiac function and structure loss due to local heart irradiation. Male Gsta4-null and wild-type mice were exposed to a single X-ray dose of 18 Gy to the heart. Six months after irradiation, immunohistochemical staining for respiratory complexes 2 and 5 indicated that radiation exposure had caused most pronounced alterations in mitochondrial morphology in Gsta4-null mice. On the other hand, wild-type mice showed a decline in cardiac function and an increase in plasma levels of troponin-I, while no such changes were observed in Gsta4-null mice. Radiation-induced Nrf2-target gene expression only in Gsta4-null mice. In conclusion, although loss of GSTA4-4 led to enhanced susceptibility of cardiac mitochondria to radiation-induced loss of morphology, cardiac function was preserved in Gsta4-null mice. We propose that this protection against cardiac function loss may occur, at least in part, by upregulation of the Nrf2 pathway. PMID:26010708

  10. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed

    Fotouhi-Ardakani, N; Batist, G

    1999-05-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  11. Biochemical studies on glutathione S-transferase from the bovine filarial worm Setaria digitata.

    PubMed

    Srinivasan, Lakshmy; Mathew, Nisha; Karunan, Twinkle; Muthuswamy, Kalyanasundaram

    2011-07-01

    Setaria digitata is a filarial worm of the cattle used as a model system for antifilarial drug screening, due to its similarity to the human filarial parasites Wuchereria bancrofti and Brugia malayi. Since filarial glutathione S-transferase (GST) is a good biochemical target for antifilarial drug development, a study has been undertaken for the biochemical characterization of GST from S. digitata. Cytosolic fraction was separated from the crude S.digitata worm homogenate by ultracentrifugation at 100,000 g and subjected to ammonium sulfate precipitation followed by affinity chromatography using GSH-agarose column. The kinetic parameters K (m) and V (max) values with respect to GSH were 0.45 mM and 0.105 μmol min(-1) mL(-1) respectively. With respect to 1-chloro-2,4-dinitrobenzene, the K (m) and V (max) values were 1.21 and 0.117 μmol min(-1) mL(-1) respectively. The effect of temperature and pH on GST enzyme activity was studied. The protein retained its enzyme activity between 0°C and 40°C, beyond which it showed a decreasing tendency, and at 80°C, the activity was lost completely. The enzyme activity was varying with change in pH, and the maximum GST activity was observed at pH 7.5. Gel filtration chromatographic studies indicated that the protein has a native molecular mass of about 54 kDa. The single band of GST subunit appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to have molecular mass of ∼27 kDa. This shows that cytosolic S. digitata GST protein is homodimeric in nature. PMID:21207063

  12. Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress.

    PubMed Central

    Raza, Haider; Robin, Marie-Anne; Fang, Ji-Kang; Avadhani, Narayan G

    2002-01-01

    The mitochondrial respiratory chain, which consumes approx. 85-90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress. PMID:12020353

  13. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    PubMed Central

    Brandauer, Josef; Vienberg, Sara G; Andersen, Marianne A; Ringholm, Stine; Risis, Steve; Larsen, Per S; Kristensen, Jonas M; Frøsig, Christian; Leick, Lotte; Fentz, Joachim; Jørgensen, Sebastian; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; Zierath, Juleen R; Goodyear, Laurie J; Pilegaard, Henriette; Treebak, Jonas T

    2013-01-01

    Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related. PMID:23918774

  14. Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2.

    PubMed

    Jemth, P; Stenberg, G; Chaga, G; Mannervik, B

    1996-05-15

    Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue. The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D. The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs. The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx. 200 mg from a 1 litre bacterial culture. The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme. The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues. These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies. 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low. The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM. The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes. The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10). PMID:8645195

  15. Biochemical Warfare on the Reef: The Role of Glutathione Transferases in Consumer Tolerance of Dietary Prostaglandins

    PubMed Central

    Whalen, Kristen E.; Lane, Amy L.; Kubanek, Julia; Hahn, Mark E.

    2010-01-01

    Background Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Principal Findings Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. Significance The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may

  16. Glutathione-S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke

    PubMed Central

    Conklin, Daniel J.; Haberzettl, Petra; Prough, Russell A.; Bhatnagar, Aruni

    2009-01-01

    Exposure to tobacco smoke impairs endothelium-dependent arterial dilation. Reactive constituents of cigarette smoke are metabolized and detoxified by glutathione-S-transferases (GSTs). Although polymorphisms in GST genes are associated with the risk of cancer in smokers, the role of these enzymes in regulating the cardiovascular effects of smoking has not been studied. The P isoform of GST (GSTP), which catalyzes the conjugation of electrophilic molecules in cigarette smoke such as acrolein, was expressed in high abundance in the mouse lung and aorta. Exposure to tobacco smoke for 3 days (5 h/day) decreased total plasma protein. These changes were exaggerated in GSTP−/− mice. Aortic rings isolated from tobacco smoke-exposed GSTP−/− mice showed greater attenuation of ACh-evoked relaxation than those from GSTP+/+ mice. The lung, plasma, and aorta of mice exposed to tobacco smoke or acrolein (for 5 h) accumulated more acrolein-adducted proteins than those tissues of mice exposed to air, indicating that exposure to tobacco smoke results in the systemic delivery of acrolein. Relative to GSTP+/+ mice, modification of some proteins by acrolein was increased in the aorta of GSTP−/− mice. Aortic rings prepared from GSTP−/− mice that inhaled acrolein (1 ppm, 5 h/day for 3 days) or those exposed to acrolein in an organ bath showed diminished ACh-induced arterial relaxation more strongly than GSTP+/+ mice. Acrolein-induced endothelial dysfunction was prevented by pretreatment of the aorta with N-acetylcysteine. These results indicate that GSTP protects against the endothelial dysfunction induced by tobacco smoke exposure and that this protection may be related to the detoxification of acrolein or other related cigarette smoke constituents. PMID:19270193

  17. Relation of Gamma-Glutamyl Transferase to Cardiovascular Events in Patients With Acute Coronary Syndromes.

    PubMed

    Ndrepepa, Gjin; Braun, Siegmund; Cassese, Salvatore; Fusaro, Massimiliano; Laugwitz, Karl-Ludwig; Schunkert, Heribert; Kastrati, Adnan

    2016-05-01

    The prognostic value of gamma-glutamyl transferase (GGT) in patients with acute coronary syndromes (ACS) has been incompletely investigated. We investigated this clinically relevant question in 2,534 consecutive patients with ACS who underwent percutaneous coronary intervention (PCI). GGT activity was measured before PCI procedure in all patients. Statin therapy at hospital discharge was prescribed in 94% of the patients. The primary outcome was 3-year mortality. Patients were divided into 3 groups: the group with GGT in the first tertile (GGT <28 U/L; n = 848 patients), the group with GGT in the second tertile (GGT 28 to <50 U/L; n = 843 patients), and the group with GGT in the third tertile (GGT ≥50 U/L; n = 843 patients). The primary outcome (all-cause deaths) occurred in 250 patients: 70 deaths (9.7%) among patients of the first, 69 deaths (9.0%) among patients of the second, and 111 deaths (14.8%) among patients of the third GGT tertile (adjusted hazard ratio [HR] 1.24, 95% CI 1.08 to 1.42, p = 0.002) and cardiac and noncardiac deaths occurred in 157 (63%) and 93 patients (37%), respectively. GGT was associated with the increased risk of noncardiac mortality (adjusted HR 1.35 [1.09 to 1.66], p = 0.005) but not cardiac mortality (adjusted HR 1.16 [0.97 to 1.38], p = 0.098; all 3 risk estimates were calculated per SD increase in the logarithmic scale of GGT activity). In conclusion, in contemporary patients with ACS treated with PCI and on statin therapy, elevated GGT activity was associated with the increased risk of all-cause and noncardiac mortality but not with the risk of cardiac mortality. PMID:26956636

  18. Glutathione S-transferases in rat olfactory epithelium: purification, molecular properties and odorant biotransformation.

    PubMed Central

    Ben-Arie, N; Khen, M; Lancet, D

    1993-01-01

    The olfactory epithelium is exposed to a variety of xenobiotic chemicals, including odorants and airborne toxic compounds. Recently, two novel, highly abundant, olfactory-specific biotransformation enzymes have been identified: cytochrome P-450olf1 and olfactory UDP-glucuronosyltransferase (UGT(olf)). The latter is a phase II biotransformation enzyme which catalyses the glucuronidation of alcohols, thiols, amines and carboxylic acids. Such covalent modification, which markedly affects lipid solubility and agonist potency, may be particularly important in the rapid termination of odorant signals. We report here the identification and characterization of a second olfactory phase II biotransformation enzyme, a glutathione S-transferase (GST). The olfactory epithelial cytosol shows the highest GST activity among the extrahepatic tissues examined. Significantly, olfactory epithelium had an activity 4-7 times higher than in other airway tissues, suggesting a role for this enzyme in chemoreception. The olfactory GST has been affinity-purified to homogeneity, and shown by h.p.l.c. and N-terminal amino acid sequencing to constitute mainly the Yb1 and Yb2 subunits, different from most other tissues that have mixtures of more enzyme classes. The identity of the olfactory enzymes was confirmed by PCR cloning and restriction enzyme analysis. Most importantly, the olfactory GSTs were found to catalyse glutathione conjugation of several odorant classes, including many unsaturated aldehydes and ketones, as well as epoxides. Together with UGT(olf), olfactory GST provides the necessary broad coverage of covalent modification capacity, which may be crucial for the acuity of the olfactory process. Images Figure 1 Figure 4 Figure 5 PMID:8503873

  19. Glutathione S-Transferase Regulation in Calanus finmarchicus Feeding on the Toxic Dinoflagellate Alexandrium fundyense

    PubMed Central

    Roncalli, Vittoria; Jungbluth, Michelle J.; Lenz, Petra H.

    2016-01-01

    The effect of the dinoflagellate, Alexandrium fundyense, on relative expression of glutathione S-transferase (GST) transcripts was examined in the copepod Calanus finmarchicus. Adult females were fed for 5-days on one of three experimental diets: control (100% Rhodomonas spp.), low dose of A. fundyense (25% by volume, 75% Rhodomonas spp.), and high dose (100% A. fundyense). Relative expression of three GST genes was measured using RT-qPCR on days 0.5, 1, 2 and 5 in two independent experiments. Differential regulation was found for the Delta and the Sigma GSTs between 0.5 to 2 days, but not on day 5 in both experiments. The third GST, a microsomal, was not differentially expressed in either treatment or day. RT-qPCR results from the two experiments were similar, even though experimental females were collected from the Gulf of Maine on different dates and their reproductive output differed. In the second experiment, expression of 39 GSTs was determined on days 2 and 5 using RNA-Seq. Global gene expression analyses agreed with the RT-qPCR results. Furthermore, the RNA-Seq measurements indicated that only four GSTs were differentially expressed under the experimental conditions, and the response was small in amplitude. In summary, the A. fundyense diet led to a rapid and transient response in C. finmarchicus in three cytosolic GSTs, while a fourth GST (Omega I) was significantly up-regulated on day 5. Although there was some regulation of GSTs in response the toxic dinoflagellate, the tolerance to A. fundyense by C. finmarchicus is not dependent on the long-term up-regulation of specific GSTs. PMID:27427938

  20. A critical perspective of the diverse roles of O-GlcNAc transferase in chromatin.

    PubMed

    Gambetta, Maria Cristina; Müller, Jürg

    2015-12-01

    O-linked β-N-Acetylglucosamine (O-GlcNAc) is a posttranslational modification that is catalyzed by O-GlcNAc transferase (Ogt) and found on a plethora of nuclear and cytosolic proteins in animals and plants. Studies in different model organisms revealed that while O-GlcNAc is required for selected processes in Caenorhabditis elegans and Drosophila, it has evolved to become required for cell viability in mice, and this has challenged investigations to identify cellular functions that critically require this modification in mammals. Nevertheless, a principal cellular process that engages O-GlcNAcylation in all of these species is the regulation of gene transcription. Here, we revisit several of the primary experimental observations that led to current models of how O-GlcNAcylation affects gene expression. In particular, we discuss the role of the stable association of Ogt with the transcription factors Hcf1 and Tet, the two main Ogt-interacting proteins in nuclei of mammalian cells. We also critically evaluate the evidence that specific residues on core histones, including serine 112 of histone 2B (H2B-S112), are O-GlcNAcylated in vivo and discuss possible physiological effects of these modifications. Finally, we review our understanding of the role of O-GlcNAcylation in Drosophila, where recent studies suggest that the developmental defects in Ogt mutants are all caused by lack of O-GlcNAcylation of a single transcriptional regulator, the Polycomb repressor protein Polyhomeotic (Ph). Collectively, this reexamination of the experimental evidence suggests that a number of recently propagated models about the role of O-GlcNAcylation in transcriptional control should be treated cautiously. PMID:25894967

  1. Controversial role of gamma-glutamyl transferase activity in cisplatin nephrotoxicity.

    PubMed

    Fliedl, Lukas; Wieser, Matthias; Manhart, Gabriele; Gerstl, Matthias P; Khan, Abdulhameed; Grillari, Johannes; Grillari-Voglauer, Regina

    2014-01-01

    Nephrotoxicity of chemotherapeutics is a major hindrance in the treatment of various tumors. Therefore, test systems that reflect mechanisms of human kidney toxicity are necessary, and to reduce animal testing cell culture based systems have to be developed. One cell type that is of specific interest in this regard are renal proximal tubular epithelial cells, as they reabsorb substances from human primary urine filtrates and thus are exposed to urinary excreted xenobiotics and are a major target of cisplatin toxicity. While animal studies using gamma glutamyl transferase (GGT) knock-out mice or GGT inhibitors show that GGT activity increases kidney toxicity of cisplatin, the use of various cell models gives contradictory results. We therefore used a cell panel of immortalized human renal proximal tubular epithelial (RPTECs) cell lines differing in GGT activity. Low GGT activity resulted in high cisplatin sensitivity, as observed in RPTEC-SV40 cells or after siRNA mediated knock-down of GGT in RPTEC/TERT1 cells that have high GGT activity. However, the addition of GGT did not rescue, but also increased cisplatin sensitivity and adding GGT inhibitor as well as substrate (glutathione) or product (cysteinyl-glycine) of GGT resulted in decreased sensitivity. While our data suggest that the use of cell panels are of value in toxicology and toxicogenomics, they also emphasize on the complex interplay of toxins with the intracellular and extracellular microenvironment. In addition, we hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation. PMID:24664430

  2. Methods for purification of glutathione transferases in the earthworm genus Eisenia, and their characterization.

    PubMed

    Borgeraas, J; Nilsen, K; Stenersen, J

    1996-06-01

    Isoenzymes of glutathione transferase (GST) were partially purified from the earthworm species Eisenia andrei and E. veneta using affinity chromatography followed by ion exchange chromatography and reversed-phase HPLC. In E. veneta, five activity peaks, named EvGST Ia, Ib, II, III and IV, were separated by anion exchange chromatography. The GSTs in E. andrei were resolved by cation exchange chromatography into six groups, named EaGST I-VI. Using reversed-phase HPLC, the affinity-purified GSTs from E. andrei and E. veneta were resolved into 14 subunits, named Ea1-Ea14 and Ev1-Ev14, respectively. EaGST I, II, IV and EvGST Ia were further characterized. These forms displayed different substrate specificity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, ethacrynic acid (ETHA) and cumene hydroperoxide, as well as different subunit composition determined by SDS-PAGE and reversed-phase HPLC. EaGST IV and EvGST Ia showed exceptionally high ETHA activity compared with the other forms. EaGST IV consisted of a homodimeric protein involving subunit Ea6 with an apparent molecular weight of 26.5 kDa, whereas EvGST Ia is composed of two different subunits (Ev9 and Ev10). Amino acid composition and N-terminal analysis of the first 33 residues of Ea6 indicated that the enzyme is most related to the pi class. Subunit Ev10 had 67% identity with Ea6, over the region sequenced (12 residues), but up to 90% identity with GSTs from several nematodes. Exposure of both species to trans-stilbene oxide, 3-methylcholanthrene and phenobarbital for three weeks did not elevate the activity of GST measured with CDNB and ETHA. PMID:8760608

  3. O-GlcNAc Transferase Directs Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension

    PubMed Central

    Barnes, Jarrod W.; Tian, Liping; Heresi, Gustavo A.; Farver, Carol F.; Asosingh, Kewal; Comhair, Suzy A. A.; Aulak, Kulwant S.; Dweik, Raed A.

    2015-01-01

    Background Idiopathic Pulmonary arterial Hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Methods and Results Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway (HBP). We measured the levels of O-linked N-acetylglucosamine modification (O-GlcNAc), O-GlcNAc transferase (OGT), and O-GlcNAc hydrolase (OGA) in control and IPAH cells and tissues. Our data suggests that the activation of the HBP directly increased OGT levels and activity triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-GlcNAc levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1 (HCF-1). Conclusions Our data demonstrate that HBP flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of HCF-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH. PMID:25663381

  4. Glutathione S Transferases Polymorphisms Are Independent Prognostic Factors in Lupus Nephritis Treated with Cyclophosphamide

    PubMed Central

    Verstuyft, Céline; Costedoat-Chalumeau, Nathalie; Hummel, Aurélie; Le Guern, Véronique; Sacré, Karim; Meyer, Olivier; Daugas, Eric; Goujard, Cécile; Sultan, Audrey; Lobbedez, Thierry; Galicier, Lionel; Pourrat, Jacques; Le Hello, Claire; Godin, Michel; Morello, Rémy; Lambert, Marc; Hachulla, Eric; Vanhille, Philippe; Queffeulou, Guillaume; Potier, Jacky; Dion, Jean-Jacques; Bataille, Pierre; Chauveau, Dominique; Moulis, Guillaume; Farge-Bancel, Dominique; Duhaut, Pierre; Saint-Marcoux, Bernadette; Deroux, Alban; Manuzak, Jennifer; Francès, Camille; Aumaitre, Olivier; Bezanahary, Holy; Becquemont, Laurent; Bienvenu, Boris

    2016-01-01

    Objective To investigate association between genetic polymorphisms of GST, CYP and renal outcome or occurrence of adverse drug reactions (ADRs) in lupus nephritis (LN) treated with cyclophosphamide (CYC). CYC, as a pro-drug, requires bioactivation through multiple hepatic cytochrome P450s and glutathione S transferases (GST). Methods We carried out a multicentric retrospective study including 70 patients with proliferative LN treated with CYC. Patients were genotyped for polymorphisms of the CYP2B6, CYP2C19, GSTP1, GSTM1 and GSTT1 genes. Complete remission (CR) was defined as proteinuria ≤0.33g/day and serum creatinine ≤124 µmol/l. Partial remission (PR) was defined as proteinuria ≤1.5g/day with a 50% decrease of the baseline proteinuria value and serum creatinine no greater than 25% above baseline. Results Most patients were women (84%) and 77% were Caucasian. The mean age at LN diagnosis was 41 ± 10 years. The frequency of patients carrying the GST null genotype GSTT1-, GSTM1-, and the Ile→105Val GSTP1 genotype were respectively 38%, 60% and 44%. In multivariate analysis, the Ile→105Val GSTP1 genotype was an independent factor of poor renal outcome (achievement of CR or PR) (OR = 5.01 95% CI [1.02–24.51]) and the sole factor that influenced occurrence of ADRs was the GSTM1 null genotype (OR = 3.34 95% CI [1.064–10.58]). No association between polymorphisms of cytochrome P450s gene and efficacy or ADRs was observed. Conclusion This study suggests that GST polymorphisms highly impact renal outcome and occurrence of ADRs related to CYC in LN patients. PMID:27002825

  5. Association of γ-glutamyl transferase with premature coronary artery disease

    PubMed Central

    GHATGE, MADANKUMAR; SHARMA, ANKIT; VANGALA, RAJANI KANTH

    2016-01-01

    Accumulating evidence from epidemiological studies suggests that higher γ-glutamyl transferase (GGT) levels in the blood are associated with the incident of cardiovascular disease (CVD), including atherosclerosis, and have prognostic importance. However, to the best of our knowledge, the association of the GGT level with premature coronary artery disease (CAD) in an Asian Indian population has not been evaluated. In the present study, 240 (120 unaffected and 120 CAD affected) young subjects (males, ≤45 years and females, ≤50 years) were selected. The markers assayed were GGT, high-sensitivity C-reactive protein, lipids, secretory phospholipase A2, neopterin, myeloperoxidase, interleukin-6, cystatin-C, tumor necrosis factor-like weak inducer of apoptosis and lipoprotein (a). The plasma GGT levels in these subjects showed a positive correlation with quantitative variables, such as waist circumference, triglycerides, neopterin levels and cross-sectional correlation with qualitative variable smoking. The findings suggest that the subjects in the highest tertile of GGT had a 2.1-fold [odds ratio (OR), 2.104; 95% confidence interval (CI), 1.063–4.165; P=0.033] higher risk of developing premature CAD in comparison with the reference tertile. Furthermore, a 1 U/l increase of GGT (on a log scale) increased the OR by 5.2-fold (OR, 5.208; 95% CI, 1.018–24.624; P=0.048) and 7.4-fold (OR, 7.492; 95% CI, 1.221–45.979; P=0.030) on addition of associated risk factors. In conclusion, the elevated plasma GGT levels potentially indicate increased oxidative stress and the risk of developing premature CAD. Therefore, these findings could be potentially used in the risk stratification of premature CAD following further evaluation. PMID:26998267

  6. Deficiency of glutathione transferase zeta causes oxidative stress and activation of antioxidant response pathways.

    PubMed

    Blackburn, Anneke C; Matthaei, Klaus I; Lim, Cindy; Taylor, Matthew C; Cappello, Jean Y; Hayes, John D; Anders, M W; Board, Philip G

    2006-02-01

    Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity. PMID:16278372

  7. Functional characterization of glutathione S-transferases associated with insecticide resistance in Tetranychus urticae.

    PubMed

    Pavlidi, Nena; Tseliou, Vasilis; Riga, Maria; Nauen, Ralf; Van Leeuwen, Thomas; Labrou, Nikolaos E; Vontas, John

    2015-06-01

    The two-spotted spider mite Tetranychus urticae is one of the most important agricultural pests world-wide. It is extremely polyphagous and develops resistance to acaricides. The overexpression of several glutathione S-transferases (GSTs) has been associated with insecticide resistance. Here, we functionally expressed and characterized three GSTs, two of the delta class (TuGSTd10, TuGSTd14) and one of the mu class (TuGSTm09), which had been previously associated with striking resistance phenotypes against abamectin and other acaricides/insecticides, by transcriptional studies. Functional analysis showed that all three GSTs were capable of catalyzing the conjugation of both 1-chloro-2,4 dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene(DCNB) to glutathione (GSH), as well as exhibiting GSH-dependent peroxidase activity toward Cumene hydroperoxide (CumOOH). The steady-state kinetics of the T. urticae GSTs for the GSH/CDNB conjugation reaction were determined and compared with other GSTs. The interaction of the three recombinant proteins with several acaricides and insecticides was also investigated. TuGSTd14 showed the highest affinity toward abamectin and a competitive type of inhibition, which suggests that the insecticide may bind to the H-site of the enzyme. The three-dimensional structure of the TuGSTd14 was predicted based on X-ray structures of delta class GSTs using molecular modeling. Structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of TuGSTd14. PMID:26047112

  8. Immunohistochemical localization and activity of glutathione transferase zeta (GSTZ1-1) in rat tissues.

    PubMed

    Lantum, Hoffman B M; Baggs, Raymond B; Krenitsky, Daria M; Board, Philip G; Anders, M W

    2002-06-01

    Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloacids, including dichloroacetic acid (DCA), and the penultimate step in the tyrosine degradation pathway. DCA is a rodent carcinogen and a common drinking water contaminant. DCA also causes multiorgan toxicity in rodents and dogs. The objective of this study was to determine the expression and activities of GSTZ1-1 in rat tissues with maleylacetone and chlorofluoroacetic acid as substrates. GSTZ1-1 protein was detected in most tissues by immunoblot analysis after immunoprecipitation of GSTZ1-1 and by immunohistochemical analysis; intense staining was observed in the liver, testis, and prostate; moderate staining was observed in the brain, heart, pancreatic islets, adrenal medulla, and the epithelial lining of the gastrointestinal tract, airways, and bladder; and sparse staining was observed in the renal juxtaglomerular regions, skeletal muscle, and peripheral nerve tissue. These patterns of expression corresponded to GSTZ1-1 activities in the different tissues with maleylacetone and chlorofluoroacetic acid as substrates. Specific activities ranged from 258 +/- 17 (liver) to 1.1 +/- 0.4 (muscle) nmol/min/mg of protein with maleylacetone as substrate and from 4.6 +/- 0.89 (liver) to 0.09 +/- 0.01 (kidney) nmol/min/mg of protein with chlorofluoroacetic acid as substrate. Rats given DCA had reduced amounts of immunoreactive GSTZ1-1 protein and activities of GSTZ1-1 in most tissues, especially in the liver. These findings indicate that the DCA-induced inactivation of GSTZ1-1 in different tissues may result in multiorgan disorders that may be associated with perturbed tyrosine metabolism. PMID:12019185

  9. Urinary π-glutathione S-transferase Predicts Advanced Acute Kidney Injury Following Cardiovascular Surgery

    PubMed Central

    Shu, Kai-Hsiang; Wang, Chih-Hsien; Wu, Che-Hsiung; Huang, Tao-Min; Wu, Pei-Chen; Lai, Chien-Heng; Tseng, Li-Jung; Tsai, Pi-Ru; Connolly, Rory; Wu, Vin-Cent

    2016-01-01

    Urinary biomarkers augment the diagnosis of acute kidney injury (AKI), with AKI after cardiovascular surgeries being a prototype of prognosis scenario. Glutathione S-transferases (GST) were evaluated as biomarkers of AKI. Urine samples were collected in 141 cardiovascular surgical patients and analyzed for urinary alpha-(α-) and pi-(π-) GSTs. The outcomes of advanced AKI (KDIGO stage 2, 3) and all-cause in-patient mortality, as composite outcome, were recorded. Areas under the receiver operator characteristic (ROC) curves and multivariate generalized additive model (GAM) were applied to predict outcomes. Thirty-eight (26.9%) patients had AKI, while 12 (8.5%) were with advanced AKI. Urinary π-GST differentiated patients with/without advanced AKI or composite outcome after surgery (p < 0.05 by generalized estimating equation). Urinary π-GST predicted advanced AKI at 3 hrs post-surgery (p = 0.033) and composite outcome (p = 0.009), while the corresponding ROC curve had AUC of 0.784 and 0.783. Using GAM, the cutoff value of 14.7 μg/L for π-GST showed the best performance to predict composite outcome. The addition of π-GST to the SOFA score improved risk stratification (total net reclassification index = 0.47). Thus, urinary π-GST levels predict advanced AKI or hospital mortality after cardiovascular surgery and improve in SOFA outcome assessment specific to AKI. PMID:27527370

  10. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

    PubMed Central

    Eudes, Aymerick; Pereira, Jose H.; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E.K.; Lee, Taek Soon; Adams, Paul D.; Keasling, Jay D.; Loqué, Dominique

    2016-01-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  11. Genetic Deficiency of Glutathione S-Transferase P Increases Myocardial Sensitivity to Ischemia-Reperfusion Injury

    PubMed Central

    Conklin, Daniel J.; Guo, Yiru; Jagatheesan, Ganapathy; Kilfoil, Peter; Haberzettl, Petra; Hill, Bradford G.; Baba, Shahid P.; Guo, Luping; Wetzelberger, Karin; Obal, Detlef; Rokosh, D. Gregg; Prough, Russell A.; Prabhu, Sumanth D.; Velayutham, Murugesan; Zweier, Jay L.; Hoetker, David; Riggs, Daniel W.; Srivastava, Sanjay; Bolli, Roberto; Bhatnagar, Aruni

    2016-01-01

    Rationale Myocardial ischemia-reperfusion (I/R) results in the generation of oxygen-derived free radicals and the accumulation of lipid peroxidation-derived unsaturated aldehydes. However, the contribution of aldehydes to myocardial I/R injury has not been assessed. Objective We tested the hypothesis that removal of aldehydes by glutathione S-transferase P (GSTP) diminishes I/R injury. Methods and Results In adult male C57BL/6 mouse hearts, Gstp1/2 was the most abundant GST transcript followed by Gsta4 and Gstm4.1, and GSTP activity was a significant fraction of the total GST activity. mGstp1/2 deletion reduced total GST activity, but no compensatory increase in GSTA and GSTM or major antioxidant enzymes was observed. Genetic deficiency of GSTP did not alter cardiac function, but in comparison with hearts from wild-type (WT) mice, the hearts isolated from GSTP-null mice were more sensitive to I/R injury. Disruption of the GSTP gene also increased infarct size after coronary occlusion in situ. Ischemia significantly increased acrolein in hearts, and GSTP deficiency induced significant deficits in the metabolism of the unsaturated aldehyde, acrolein, but not in the metabolism 4-hydroxy-trans-2-nonenal (HNE) or trans-2-hexanal; and, upon ischemia, the GSTP-null hearts accumulated more acrolein-modified proteins than WT hearts. GSTP-deficiency did not affect I/R-induced free radical generation, JNK activation or depletion of reduced glutathione. Acrolein-exposure induced a hyperpolarizing shift in INa, and acrolein-induced cell death was delayed by SN-6, a Na+/Ca++ exchange inhibitor. Cardiomyocytes isolated from GSTP-null hearts were more sensitive than WT myocytes to acrolein-induced protein crosslinking and cell death. Conclusions GSTP protects the heart from I/R injury by facilitating the detoxification of cytotoxic aldehydes such as acrolein. PMID:26169370

  12. Expression of glutathione, glutathione peroxidase and glutathione S-transferase pi in canine mammary tumors

    PubMed Central

    2014-01-01

    Background Glutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy. Results The immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death. The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors. The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes. Conclusion Therefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in

  13. Inhibition characteristics of hypericin on rat small intestine glutathione-S-transferases.

    PubMed

    Tuna, Gamze; Kulaksiz Erkmen, Gulnihal; Dalmizrak, Ozlem; Dogan, Arin; Ogus, I Hamdi; Ozer, Nazmi

    2010-10-01

    Glutathione-S-transferases constitute a family of enzymes involving in the detoxification of xenobiotics, signalling cascades and serving as ligandins or/and catalyzing the conjugation of various chemicals and drugs. The widely expressed cytosolic GST-pi is a marker protein in various cancers and its increased concentration is linked to drug resistance. GST-pi is autoregulated by S-glutathionylation and it catalyzes the S-glutathionylation of other proteins in response to oxidative or nitrosative stress. S-glutathionylation of GST-pi results in multimer formation and the breakage of ligand binding interactions with c-Jun NH(2)-terminal kinase (JNK). Another widely expressed GST enzyme, GST-alpha is assumed as a marker in hepatocellular damage, is implicated in cancer, asthma, cardiovascular disease and response to chemotherapy. Although, it was shown that hypericin binds and inhibits GST-alpha and GST-pi, the inhibition characteristics have not been investigated in detail. The aim of this study was to investigate the effects of hypericin on major GSTs; GST-alpha and GST-pi purified from rat small intestine. When GSH used as varied substrate the inhibition pattern with hypericin was uncompetitive for GST-alpha (K(i)=0.16 + or - 0.02 microM) and noncompetitive for GST-pi (K(i) = 2.46 + or - 0.43 microM). While using CDNB (1-chloro-2,4-dinitrobenzene) as the varied substrate, the inhibition patterns were noncompetitive for GST-alpha and competitive for GST-pi; K(i) values for GST-alpha and GST-pi were 1.91 + or - 0.21 and 0.55 + or - 0.07 microM, respectively. Since hypericin accumulated in cancer cells and important in photodynamic therapy (PDT), inhibition of GST-alpha and GST-pi by hypericin might increase the effectivity of the treatment. Considering that GST-pi is responsible for the drug resistance its inhibition might increase the benefit obtained from chemotherapy. PMID:20637187

  14. Detection and adequacy evaluation of erythrocyte glutathione transferase on levels of circulating toxins in hemodialysis patients.

    PubMed

    Yin, Rui; Qiu, Hui; Zuo, Huaiyun; Cui, Min; Zhai, Nailiang; Zheng, Hongguang; Zhang, Dewei; Huo, Ping; Hong, Min

    2016-08-01

    To explore detection and adequacy evaluation of erythrocyte glutathione S transferase (GST) on levels of circulating toxins in hemodialysis patients in Qinhuangdao region in China, this study divided 84 cases of long-term, end-stage hemodialysis patients into 2 groups: one group of 33 cases of adequate hemodialysis (spKt/V ≥ 1.3) and another group of 51 cases of inadequate hemodialysis (spKt/V < 1.3), according to the urea index value of the unit chamber model (spKt/V). Another 50 cases of subjects found healthy by a physical examination were taken as the control group, and the differences in the related clinical and biochemical indexes of the 3 groups were compared and analyzed. The levels of GST, creatinine, high sensitivity C-reactive protein (hs-CRP), transferrin saturation (TSAT), parathyroid hormone (PTH), interleukin-2,6,8 (IL-2,6,8) and tumor necrosis factor-a (TNF-a) in the hemodialysis group were significantly higher than those in the control group (P < 0.05), and GST, IL-2, 6, 8, and TNF-a levels in the inadequate hemodialysis group were significantly higher than in the adequate hemodialysis group (P < 0.05). Pearson's relevant analysis showed that the levels of GST and spKt/V, IL-2, IL-6, IL-8, and TNF-a have a positive correlation (P < 0.05), and they have no correlation with levels of creatinine, hs-CRP, TSAT, and PHT (P > 0.05). There were 23 patients with levels of spKt/V ≥ 1.3 after adjusting the dialysis solution for 51 cases of inadequate hemodialysis patients, and the GST level after the adjustment was significantly lower than that before the adjustment, but still higher than that in the adequate dialysis group. This concludes that the maintenance of hemodialysis in patients has certain relevance on spKt/V and associated inflammatory factors. Through the study, it can be determined that GST can effectively respond to adequate hemodialysis, which has a guiding significance on adjusting the blood dialysis solution in clinical practice. PMID

  15. Habitual consumption of fruits and vegetables: associations with human rectal glutathione S-transferase.

    PubMed

    Wark, Petra A; Grubben, Marina J A L; Peters, Wilbert H M; Nagengast, Fokko M; Kampman, Ellen; Kok, Frans J; van 't Veer, Pieter

    2004-11-01

    The glutathione (GSH)/glutathione S-transferase (GST) system is an important detoxification system in the gastrointestinal tract. A high activity of this system may benefit cancer prevention. The aim of the study was to assess whether habitual consumption of fruits and vegetables, especially citrus fruits and brassica and allium vegetables, is positively associated with parameters reflecting the activity of the GSH/GST enzyme system in human rectal mucosa. GST enzyme activity, GST isoenzyme levels of GST-alpha (A1-1, A1-2 and A2-2), -mu (M1-1) and -pi (P1-1), and GSH levels were measured in rectal biopsies from 94 subjects. Diet, lifestyle, GSTM1 and GSTT1 null polymorphisms were assessed. Mean GST enzyme activity was 237 nmol/min/mg protein (SD = 79). Consumption of citrus fruits was positively associated with GST enzyme activity [difference between high and low consumption: 28.9 (95% confidence interval (CI) = 9.3-48.6) nmol/min/mg protein], but was not associated with the other parameters. A positive association with brassica vegetables was found among carriers of the GSTM1-plus genotype [difference between high and low consumption: 22.6 (95% CI = 0.2-45.0) nmol/min/mg protein], but not among GSTM1-null individuals (-25.8 nmol/min/mg protein, 95% CI = -63.3-11.8). This is in line with a positive association between consumption of brassica vegetables and GSTM isoenzyme level [difference between high and low consumption: 67.5%, 95% CI = (6.8-162.7)]. Consumption of allium vegetables was not associated with GST enzyme activity, but negatively with GSTP1-1 levels [difference between high and low consumption: -23.3%, 95% CI = (-35.5; -8.6)]. Associations were similar among those with the GSTT1-plus and GSTT1-null genotype. In conclusion, variations in habitual consumption of fruits, particularly citrus fruits, and of vegetables, in particular brassica vegetables, among those with the GSTM1-plus genotype, may contribute to variations in human rectal GST enzyme

  16. Proteomic and immunochemical characterization of glutathione transferase as a new allergen of the nematode Ascaris lumbricoides.

    PubMed

    Acevedo, Nathalie; Mohr, Jens; Zakzuk, Josefina; Samonig, Martin; Briza, Peter; Erler, Anja; Pomés, Anna; Huber, Christian G; Ferreira, Fatima; Caraballo, Luis

    2013-01-01

    Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments. PMID:24223794

  17. Prognostic significance of glutathione S-transferase-pi in invasive breast cancer.

    PubMed

    Huang, Jingxiang; Tan, Puay-Hoon; Thiyagarajan, Jayabaskar; Bay, Boon-Huat

    2003-06-01

    Glutathione S-transferase pi (GST-pi), a Phase II detoxification enzyme, has recently been implicated in protection against apoptosis. Expression of GST-pi and Bcl-2 protein, an established apoptosis marker, was analyzed by immunohistochemistry in 116 cases of infiltrative ductal breast carcinomas in Singapore women. The markers were correlated with apoptosis detected by the TUNEL method and clinico-pathological parameters. There were 67 (58%) GST-pi-positive breast tumors and 43 (37%) Bcl-2-positive tumors. In a large proportion of GST-pi-positive/Bcl-2-positive tumors, there was a distinct accumulation of the GST-pi enzyme within the nucleus of cancer cells when examined by double immunofluorescence labeling under confocal microscopy. GST-pi immunoreactivity was not significantly correlated with any of the traditional histologic factors known to influence prognosis, whereas Bcl-2 overexpression was associated with reduced size of primary tumor (P =.021) and positive estrogen receptor status (P =.001). Univariate analysis revealed that GST-pi-positive, Bcl-2-positive, and lower histological grade tumors had decreased levels of apoptosis (P =.024, P =.011, and P =.029, respectively). However, multivariate analysis showed that histological grade and Bcl-2, but not GST-pi, immunoreactivity were correlated with apoptotic status. The Kaplan-Meier disease-free survival curves showed a significant difference between GST-pi-positive and GST-pi-negative breast cancer cases (P =.002). Disease-free survival in patients with GST-pi-positive tumors was also worse than that in patients with GST-pi-negative tumors in the group who had adjuvant chemotherapy (P =.04). In patients who were lymph node positive, GST-pi immunopositivity was found to influence disease-free survival. Recurrence of tumors was also significantly affected by GST-pi immunoreactivity (relative risk of 8.1). The findings indicate that GST-pi-positive tumors are more aggressive and have a poorer prognosis than

  18. Infection with Salmonella typhimurium modulates the immune response to Schistosoma mansoni glutathione-S-transferase.

    PubMed Central

    Comoy, E E; Vendeville, C; Capron, A; Thyphronitis, G

    1997-01-01

    Immune response polarization is controlled by several factors, including cytokines, antigen-presenting cells, antigen dose, and others. We have previously shown that adjuvants and live vectors play a critical role in polarization. Thus, immunization with the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28-GST) in aluminum hydroxide induced a type 2 cytokine profile and the production of immunoglobulin G1 (IgG1)- and IgE-specific antibodies. In contrast, mice infected with recombinant Salmonella typhimurium expressing Sm28-GST developed a type 1 cytokine profile and produced IgG2a-specific antibodies against Sm28-GST and Salmonella antigens. In this study, to determine if S. typhimurium not expressing Sm28-GST would still influence the type of the response against this antigen, we compared the profiles of the immune responses generated against Sm28-GST administered in alum in mice infected and not infected with S. typhimurium. Infected mice generated both IgG1 and IgG2a antibodies against Sm28-GST, while noninfected mice produced only IgG1 anti-Sm28-GST antibodies. Moreover, interleukin-4 (IL-4) mRNA expression in infected mice was near background levels, while gamma interferon (IFN-gamma) mRNA expression in coinfected mice was significantly higher than in mice immunized with Sm28-GST in alum only. However, after antigen-specific stimulation in vitro with Sm28-GST, levels of IL-4 and IFN-gamma cytokine production were similar in the two groups of mice. These results suggest that (i) the immune milieu produced during an infection may modify the response against an irrelevant antigen and (ii) isotype switching may be influenced by the cytokine environment of a bystander immune response, even though the specific antigen-driven cytokine production is not modified. Thus, the isotypic profile is not always an absolute reflection of the cytokines produced by antigen-specific Th cells. PMID:9234784

  19. Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells.

    PubMed

    van Gisbergen, Marike W; Cebula, Marcus; Zhang, Jie; Ottosson-Wadlund, Astrid; Dubois, Ludwig; Lambin, Philippe; Tew, Kenneth D; Townsend, Danyelle M; Haenen, Guido R M M; Drittij-Reijnders, Marie-José; Saneyoshi, Hisao; Araki, Mika; Shishido, Yuko; Ito, Yoshihiro; Arnér, Elias S J; Abe, Hiroshi; Morgenstern, Ralf; Johansson, Katarina

    2016-06-01

    Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance

  20. Steady-state kinetics and chemical mechanism of octopus hepatopancreatic glutathione transferase.

    PubMed Central

    Tang, S S; Chang, G G

    1995-01-01

    The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid ([2,3-dichloro-4-(2-methyl-enebutyryl) phenoxy]acetic acid) binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p2H) = 8.011, in which kcat. was independent of pH or p2H, the solvent isotope effects on V and V/KmGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/KmCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pKa of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pKa of glutathione in aqueous solution. This lowering of the pKa value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr7, which had a pKa value of 8.46 +/- 0.09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction. Images Scheme 2 PMID:7619078

  1. In vitro kinetics of hepatic glutathione s-transferase conjugation in largemouth bass and brown bullheads

    SciTech Connect

    Gallagher, E.P.; Sheehy, K.M.; Lame, M.W.; Segall, H.J.

    2000-02-01

    The kinetics of glutathione 5-transferase (GST) catalysis were investigated in largemouth bass (Micropterus salmoides) and brown bullheads (Amerius nebulosus), two freshwater fish species found in a variety of polluted waterways in the eastern US. The initial rates of hepatic GST activity toward four GST substrates, including 1-chloro-2,4-dinitrobenzene, ethacrynic acid, {Delta}5-androstene-17-dione, and nitrobutyl chloride, were significantly higher in brown bullheads than in largemouth bass. Hepatic GST activity toward 1,2-dichloro-4-nitrobenzene, a {mu}-class GST substrate in rodents, was not detectable in either species. Liver cytosolic GSTs were more efficient in bullheads than in bass at catalyzing 1-chloro-2,4-dinitrobenzene-reduced glutathione (CDNB-GSH) conjugation over a broad range of electrophile (CDNB) concentrations, including those representative of environmental exposure. In contrast, largemouth bass maintained higher ambient concentrations of GSH, the nucleophilic cofactor for GST-mediated conjugation, than brown bullheads. Biphasic kinetics for GST-CDNB conjugation under conditions of variable GSH concentration were apparent in Eadie-Hofstee plots of the kinetic data, suggesting the presence of at least two hepatic GST isozymes with markedly different K{sub m} values for GSH in both species. The GST-CDNB reaction rate data obtained under conditions of variable GSH were well fitted (R{sup 2} = 0.999) by the two-enzyme Michaelis-Menten equation. In addition, Western blotting experiments confirmed the presence of two different hepatic GST-like proteins in both largemouth bass and brown bullhead liver. Collectively, these findings indicate that largemouth bass and brown bullhead GSTs catalyze the conjugation of structurally diverse, class-specific GST substrates, and that brown bullheads exhibit higher initial rates of GST activity than largemouth bass. The relatively higher rates of in vitro liver GST activity at the low substrate concentrations

  2. Glutathione S-transferase activity in follicular fluid from women undergoing ovarian stimulation: role in maturation.

    PubMed

    Meijide, Susana; Hernández, M Luisa; Navarro, Rosaura; Larreategui, Zaloa; Ferrando, Marcos; Ruiz-Sanz, José Ignacio; Ruiz-Larrea, M Begoña

    2014-10-01

    Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educación, Universiades e Investigación, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01). PMID:26461371

  3. Catalytic characterization of human microsomal glutathione S-transferase 2: identification of rate-limiting steps.

    PubMed

    Ahmad, Shabbir; Niegowski, Damian; Wetterholm, Anders; Haeggström, Jesper Z; Morgenstern, Ralf; Rinaldo-Matthis, Agnes

    2013-03-12

    Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 μM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of ∼0.2 μmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S

  4. Proteomic and Immunochemical Characterization of Glutathione Transferase as a New Allergen of the Nematode Ascaris lumbricoides

    PubMed Central

    Acevedo, Nathalie; Mohr, Jens; Zakzuk, Josefina; Samonig, Martin; Briza, Peter; Erler, Anja; Pomés, Anna; Huber, Christian G.; Ferreira, Fatima; Caraballo, Luis

    2013-01-01

    Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments. PMID:24223794

  5. O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

    SciTech Connect

    Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika; Nomura, Tomoko; Ito, Makiko; Nishio, Yosuke; Nadano, Daita; Matsuda, Tsukasa; Furukawa, Koichi; Okajima, Tetsuya

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer We characterized A130022J15Rik (Eogt1)-a mouse gene homologous to Drosophila Eogt. Black-Right-Pointing-Pointer Eogt1 encodes EGF domain O-GlcNAc transferase. Black-Right-Pointing-Pointer Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. Black-Right-Pointing-Pointer O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.

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

    PubMed

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

    2016-06-01

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

  7. Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro

    PubMed Central

    Trombley, Michael P.; Post, Deborah M. B.; Rinker, Sherri D.; Reinders, Lorri M.; Fortney, Kate R.; Zwickl, Beth W.; Janowicz, Diane M.; Baye, Fitsum M.; Katz, Barry P.; Spinola, Stanley M.; Bauer, Margaret E.

    2015-01-01

    Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis. PMID:25902140

  8. Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite.

    PubMed

    Santos, Jorge M; Duarte, Neuza; Kehrer, Jessica; Ramesar, Jai; Avramut, M Cristina; Koster, Abraham J; Dessens, Johannes T; Frischknecht, Friedrich; Chevalley-Maurel, Séverine; Janse, Chris J; Franke-Fayard, Blandine; Mair, Gunnar R

    2016-06-28

    Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid-a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony-is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission. PMID:27303037

  9. Glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured rat hepatocytes treated with tocotrienol and tocopherol.

    PubMed

    Ong, F B; Wan Ngah, W Z; Shamaan, N A; Md Top, A G; Marzuki, A; Khalid, A K

    1993-09-01

    1. The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated. 2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1-2 days. 3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1-3 days. 4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 microM tocotrienol at 1-3 days in culture compared to the control. PMID:7903615

  10. The molecular basis for the post-translational addition of amino acids by L/F transferase in the N-end rule pathway.

    PubMed

    Fung, Angela Wai S; Fahlman, Richard P

    2015-01-01

    The N-end rule pathway is a conserved targeted proteolytic process observed in organisms ranging from eubacteria to mammals. The N-end rule relates the metabolic stability of a protein to its N-terminal amino acid residue. The identity of the N-terminal amino acid residue is a primary degradation signal, often referred to as an N-degron, which is recognized by the components of the N-end rule when it is a destabilizing N-terminus. N-degrons may be exposed by non-processive proteolytic cleavages or by post-translational modifications. One modification is the post-translational addition of amino acids to the N-termini of proteins, a reaction catalyzed by aminoacyl-tRNA protein transferases. The aminoacyl-tRNA protein transferase in eubacteria like Escherichia coli is L/F transferase. Recent investigations have reported unexpected observations regarding the L/F transferase catalytic mechanism and its mechanisms of substrate recognition. Additionally, recent proteome-wide identification of putative in vivo substrates facilitates hypothesis into the yet elusive biological functions of the prokaryotic N-end rule pathway. Here we summarize the recent findings on the molecular mechanisms of catalysis and substrate recognition by the E. coli L/F transferase in the prokaryotic N-end rule pathway. PMID:25692952

  11. Isolation and identification of kahweol palmitate and cafestol palmitate as active constituents of green coffee beans that enhance glutathione S-transferase activity in the mouse.

    PubMed

    Lam, L K; Sparnins, V L; Wattenberg, L W

    1982-04-01

    Glutathione (GSH) S-transferase is a major detoxification enzyme system that catalyzes the binding of a variety of electrophiles, including reactive forms of chemical carcinogens, to GSH. Green coffee beans fed in the diet induced increased GSH S-transferase activity in the mucosa of the small intestine and in the liver of mice. A potent compound that induces increased GSH S-transferase activity was isolated from green coffee beans and identified as kahweol palmitate. The corresponding free alcohol, kahweol, and its synthetic monoacetate are also potent inducers of the activity of GSH S-transferase. A similar diterpene ester, cafestol palmitate, isolated from green coffee beans was active but less so than was kahweol palmitate. Likewise, the corresponding alcohol, cafestol, and its monoacetate showed moderate potency as inducers of increased GSH S-transferase activity. Kahweol palmitate and cafestol palmitate were extracted from green coffee beans into petroleum ether. The petroleum ether extract was fractionated by preparative normal-phase and reverse-phase liquid chromatographies successively. Final purification with silver nitrate-impregnated thin-layer chromatography yielded the pure palmitates of cafestol and kahweol. The structures were determined by examination of the spectroscopic data of the esters and their parent alcohols and by derivative comparison. PMID:7059995

  12. Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes.

    PubMed Central

    Zucker, S D; Goessling, W; Ransil, B J; Gollan, J L

    1995-01-01

    To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell. Images PMID:7560084

  13. Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid1[W

    PubMed Central

    Sonnante, Gabriella; D'Amore, Rosalinda; Blanco, Emanuela; Pierri, Ciro L.; De Palma, Monica; Luo, Jie; Tucci, Marina; Martin, Cathie

    2010-01-01

    Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes. PMID:20431089

  14. Effect of municipal waste water effluent upon the expression of Glutathione S-transferase isoenzymes of brine shrimp Artemia.

    PubMed

    Grammou, Athina; Papadimitriou, Chrisa; Samaras, Peter; Vasara, Eleni; Papadopoulos, Athanasios I

    2011-06-01

    Multiple isoenzymes of the detoxification enzyme family Glutathione S-transferase are expressed in the brine shrimp Artemia. The number of the major ones detected in crude extract by means of chromatofocusing varied between three and four, depending on the age. Two isoenzymes, one alkaline and one neutral (with corresponding isoelectric points of 8.5 and 7.2) appear to be dominant in all three developmental stages studied, (24, 48, and 72 h after hatching). Culturing Artemia for 48 h after hatching, in artificial sea water prepared by municipal wastewater effluent resulted to significant alterations of the isoenzyme profile. In comparison to organisms cultured for the same period of time in artificial sea water prepared by filtered tap water, the expression of the alkaline isoenzyme decreased by 62% while that of the neutral isoenzyme increased by 58%. Furthermore, the enzyme activity of the major isoenzyme of the acidic area increased by more than two folds. It is worth mentioning that although the specific activity of the total enzyme in the whole body homogenate was elevated, no statistically significant alteration of the Km value was observed. These findings suggest that study of the isoenzyme profile of Glutathione S-transferase may offer high sensitivity in detecting environmental pollution and needs to be further investigated. PMID:21429555

  15. Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner.

    PubMed

    Khan, Rashid Ahmed; Liu, Ji Yuan; Rashid, Maryam; Wang, Dun; Zhang, Ya Lin

    2013-01-01

    Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity. PMID:23528854

  16. Conversion of melphalan to 4-(glutathionyl)phenylalanine. A novel mechanism for conjugation by glutathione-S-transferases.

    PubMed

    Dulik, D M; Fenselau, C

    1987-01-01

    One of the conjugates of melphalan, characterized following incubation with glutathione (GSH) and immobilized microsomal glutathione-S-transferases, has been identified as 4-(glutathionyl)-phenylalanine. This conjugate is formed by displacement of the mustard moiety. The structure was confirmed by reaction of the corresponding 4-halophenylalanines with GSH as well as by TLC, HPLC, and FAB mass spectrometry. Evidence is presented here to support the hypothesis that this novel reaction occurs via a cyclic aziridinium ion. To test this proposed mechanism, N,N-dimethyl-p-toluidine and its corresponding quaternary ammonium iodide salt were incubated with GSH in the presence of immobilized glutathione-S-transferases at 37 degrees C for 1 hr at pH 7.4. The tertiary amine did not react, whereas the quaternary compound produced 4-(glutathionyl)toluene. The effect of ring substituent requirements for the reaction was evaluated. The formation of GSH adducts of alkylating agents may be a factor in the development of resistance to these drugs. PMID:2882977

  17. Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner

    PubMed Central

    Khan, Rashid Ahmed; Liu, Ji Yuan; Rashid, Maryam; Wang, Dun; Zhang, Ya Lin

    2013-01-01

    Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity. PMID:23528854

  18. A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

    PubMed Central

    Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

    2010-01-01

    Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

  19. Structure and expression of a cluster of glutathione S-transferase genes from a marine fish, the plaice (Pleuronectes platessa).

    PubMed Central

    Leaver, M J; Wright, J; George, S G

    1997-01-01

    Glutathione S-transferases are involved in the detoxification of reactive electrophilic compounds, including intracellular metabolites, drugs, pollutants and pesticides. A cluster of three glutathione S-transferase genes, designated GSTA, GSTA1 and GSTA2, was isolated from the marine flatfish, plaice (Pleuronectes platessa). GSTA and GSTA1 code for protein products with 76% amino acid identity. GSTA2 appears to contain a single nucleotide deletion which would render any product non-functional. All of these genes consist of six exons of similar sizes and greater than 70% nucleotide identity, and are interrupted by five introns of differing sizes. GSTA and GSTA1 mRNAs were present in a range of tissues, while GSTA2 mRNA was no detected. Expression of GSTA mRNA was increased in plaice intestine and spleen by pretreatment with beta-naphthoflavone, and expression of both GSTA and GSTA1 mRNAs was increased in plaice liver and gill by pretreatment with the peroxisome proliferating agent perfluoro-octanoic acid. PMID:9020873

  20. Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer

    PubMed Central

    Zonetti, Maria Josè; Fisco, Tommaso; Polidoro, Chiara; Bocchinfuso, Gianfranco; Palleschi, Antonio; Novelli, Giuseppe; Spagnoli, Luigi G.

    2016-01-01

    Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn't retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors. PMID:26799588

  1. Mitogen-activated protein kinase p38b interaction with delta class glutathione transferases from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, Jeerang; Sukittikul, Suchada; Saisawang, Chonticha; Ketterman, Albert J

    2012-01-01

    Glutathione transferases (GSTs) are a family of multifunctional enzymes involved in xenobiotic biotransformation, drug metabolism, and protection against oxidative damage. The p38b mitogen-activated protein kinase is involved in cellular stress response. This study screened interactions between Drosophila melanogaster Meigen (Diptera: Drosophilidae) Delta class glutathione transferases (DmGSTs) and the D. melanogaster p38b MAPK. Therefore, 12 DmGSTs and p38b kinase were obtained as recombinant proteins. The study showed that DmGSTD8 and DmGSTD11b significantly increased p38b activity toward ATF2 and jun, which are transcription factor substrates. DmGSTD3 and DmGSTD5 moderately increased p38b activity for jun. In addition, GST activity in the presence of p38b was also measured. It was found that p38b affected substrate specificity toward CDNB (1-chloro-2,4-dinitrobenzene) and DCNB (1,2-dichloro-4-nitrobenzene) of several GST isoforms, i.e., DmGSTD2, DmGSTD5, DmGSTD8, and DmGSTD11b. The interaction of a GST and p38b can affect the substrate specificity of either enzyme, which suggests induced conformational changes affecting catalysis. Similar interactions do not occur for all the Delta enzymes and p38b, which suggests that these interactions could be specific. PMID:23438069

  2. Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni

    PubMed Central

    Fage, Christopher D.; Brown, Dusty B.; Boll, Joseph M.; Keatinge-Clay, Adrian T.; Trent, M. Stephen

    2014-01-01

    The foodborne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a soluble sulfatase-like catalytic domain in the periplasm. The atomic structure of the catalytic domain of EptC (cEptC) was crystallized and solved to a resolution of 2.40 Å. cEptC adopts the α/β/α fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr266 residue was observed that was hypothesized to mimic a covalent pEtN–enzyme intermediate. The requirement for Thr266 as well as the nearby residues Asn308, Ser309, His358 and His440 was ascertained via in vivo activity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors. PMID:25286856

  3. Aniline exposure associated with up-regulated transcriptional responses of three glutathione S-transferase Delta genes in Drosophila melanogaster.

    PubMed

    Chan, Wen-Chiao; Chien, Yi-Chih; Chien, Cheng-I

    2015-03-01

    Complex transcriptional profile of glutathione S-transferase Delta cluster genes occurred in the developmental process of the fruit fly Drosophila melanogaster. The purpose of this project was to quantify the expression levels of Gst Delta class genes altered by aniline exposure and to understand the relationship between aniline dosages and the variation of Gst Delta genes expressed in D. melanogaster. Using RT-PCR expression assays, the expression patterns of the transcript mRNAs of the glutathione S-transferase Delta genes were revealed and their expression levels were measured at eggs, larvae, pupae and adults. The adult stage was selected for further dose-response assays. After analysis, the results indicated that three Gst Delta genes (Gst D2, Gst D5 and Gst D6) were found to show a peak of up-regulated transcriptional response at 6-8h of exposure of aniline. Furthermore, the dose-response relationship of their induction levels within the dose regiments (from 1.2 to 2.0 μl/tube) had been measured. The expression patterns and annotations of these genes were discussed in the context. PMID:25682008

  4. Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors.

    PubMed Central

    McLellan, L I; Judah, D J; Neal, G E; Hayes, J D

    1994-01-01

    Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic

  5. Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Alber, Birgit E; Fuchs, Georg

    2006-09-01

    The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route. PMID:16952935

  6. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

    PubMed

    Wongsantichon, Jantana; Robinson, Robert C; Ketterman, Albert J

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  7. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

    PubMed Central

    Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  8. S-Glutathionylation of Keap1: a new role for glutathione S-transferase pi in neuronal protection.

    PubMed

    Carvalho, Andreia Neves; Marques, Carla; Guedes, Rita C; Castro-Caldas, Margarida; Rodrigues, Elsa; van Horssen, Jack; Gama, Maria João

    2016-05-01

    Oxidative stress is a key pathological feature of Parkinson's disease (PD). Glutathione S-transferase pi (GSTP) is a neuroprotective antioxidant enzyme regulated at the transcriptional level by the antioxidant master regulator nuclear factor-erythroid 2-related factor 2 (Nrf2). Here, we show for the first time that upon MPTP-induced oxidative stress, GSTP potentiates S-glutathionylation of Kelch-like ECH-associated protein 1 (Keap1), an endogenous repressor of Nrf2, in vivo. S-glutathionylation of Keap1 leads to Nrf2 activation and subsequently increases expression of GSTP. This positive feedback regulatory loop represents a novel mechanism by which GSTP elicits antioxidant protection in the brain. PMID:27086966

  9. Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars exposed to dietary cadmium.

    PubMed

    Vlahović, Milena; Ilijin, Larisa; Mrdaković, Marija; Todorović, Dajana; Matić, Dragana; Lazarević, Jelica; Mataruga, Vesna Perić

    2016-06-01

    Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification. PMID:27084993

  10. Glutathione S-transferase (GST) genes in the red flour beetle, Tribolium castaneum, and comparative analysis with five additional insects.

    PubMed

    Shi, Houxia; Pei, Lianghong; Gu, Shasha; Zhu, Shicheng; Wang, Yanyun; Zhang, Yi; Li, Bin

    2012-11-01

    Glutathione S-transferases are important detoxification enzymes involved in insecticide resistance. Sequencing the Tribolium castaneum genome provides an opportunity to investigate the structure, function, and evolution of GSTs on a genome-wide scale. Thirty-six putative cytosolic GSTs and 5 microsomal GSTs have been identified in T. castaneum. Furthermore, 40, 35, 13, 23, and 32 GSTs have been discovered the other insects, Drosophila, Anopheles, Apis, Bombyx, and Acyrthosiphon, respectively. Phylogenetic analyses reveal that insect-specific GSTs, Epsilon and Delta, are the largest species-specific expanded GSTs. In T. castaneum, most GSTs are tandemly arranged in three chromosomes. Particularly, Epsilon GSTs have an inverted long-fragment duplication in the genome. Other four widely distributed classes are highly conserved in all species. Given that GSTs specially expanded in Tribolium castaneum, these genes might help to resist poisonous chemical environments and produce resistance to kinds of different insecticides. PMID:22824654

  11. Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.

    PubMed

    Kim, Eun J; Abramowitz, Lara K; Bond, Michelle R; Love, Dona C; Kang, Dong W; Leucke, Hans F; Kang, Dae W; Ahn, Jong-Seog; Hanover, John A

    2014-06-18

    The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants. PMID:24866374

  12. The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins.

    PubMed

    Wang, Yue-Yue; Luo, Hong-Dou; Zhang, Xiao-Sheng; Lin, Tao; Jiang, Hui; Li, Yong-Quan

    2016-03-01

    Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of acyl carrier proteins (ACPs) in fatty acid synthases (FASs), ACPs in polyketide synthases, and peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) in all organisms. Some bacterial PPTases have broad substrate specificities for ACPs/PCPs and/or coenzyme A (CoA)/CoA analogs, facilitating their application in metabolite production in hosts and/or labeling of ACPs/PCPs, respectively. Here, a group II PPTase SchPPT from Streptomyces chattanoogensis L10 was characterized to accept a heterologous ACP and acetyl-CoA. Thus, SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of ACPs. PMID:26748983

  13. Transcriptome-wide identification and expression analysis of glutathione S-transferase genes involved in flavonoids accumulation in Dracaena cambodiana.

    PubMed

    Zhu, Jia-Hong; Li, Hui-Liang; Guo, Dong; Wang, Ying; Dai, Hao-Fu; Mei, Wen-Li; Peng, Shi-Qing

    2016-07-01

    Dragon's blood is a traditional medicine widely used in the world, and the main components of which are flavonoids. However, little is known about its formation mechanism. Previous studies indicate that plant glutathione S-transferase (GST) genes are involved in transportation of flavonoids from cytosolic synthesis to vacuolar accumulation. In this study, 20 Dracaena cambodiana GST genes (DcGSTs) were identified based on transcriptome database. Phylogenetic analysis revealed that 20 DcGSTs belonged to seven different classes. Tissue-specific expression analysis suggested that DcGSTs displayed differential expressions either in their transcript abundance or expression patterns under normal growth conditions. The transcript profiles of three DcGSTs in response to the inducer of dragon's blood were strongly correlated with flavonoids biosynthetic genes, consistent with dragon's blood accumulation. Our survey provides useful information for future studies on GST genes involved in dragon's blood formation in D. cambodiana. PMID:27208821

  14. Micro-Plasticity of Genomes As Illustrated by the Evolution of Glutathione Transferases in 12 Drosophila Species

    PubMed Central

    Saisawang, Chonticha; Ketterman, Albert J.

    2014-01-01

    Glutathione transferases (GST) are an ancient superfamily comprising a large number of paralogous proteins in a single organism. This multiplicity of GSTs has allowed the copies to diverge for neofunctionalization with proposed roles ranging from detoxication and oxidative stress response to involvement in signal transduction cascades. We performed a comparative genomic analysis using FlyBase annotations and Drosophila melanogaster GST sequences as templates to further annotate the GST orthologs in the 12 Drosophila sequenced genomes. We found that GST genes in the Drosophila subgenera have undergone repeated local duplications followed by transposition, inversion, and micro-rearrangements of these copies. The colinearity and orientations of the orthologous GST genes appear to be unique in many of the species which suggests that genomic rearrangement events have occurred multiple times during speciation. The high micro-plasticity of the genomes appears to have a functional contribution utilized for evolution of this gene family. PMID:25310450

  15. The Role of Glutathione S-transferase P in signaling pathways and S-glutathionylation in Cancer

    PubMed Central

    Tew, Kenneth D.; Manevich, Yefim; Grek, Christina; Xiong, Ying; Uys, Joachim; Townsend, Danyelle M.

    2011-01-01

    Glutathione S-transferase P is abundantly expressed in some mammalian tissues, particularly those associated with malignancies. While the enzyme can catalyze thioether bond formation between some electrophilic chemicals and GSH, novel non-detoxification functions are now ascribed to it. This review summarizes recent material that implicates GSTP in mediating S-glutathionylation of specific clusters of target proteins and in reactions that define a negative regulatory role in some kinase pathways through ligand or protein:protein interactions. It is becoming apparent that GSTP participates in the maintenance of cellular redox homeostasis through a number of convergent and divergent mechanisms. Moreover, drug platforms that have GSTP as a target have produced some interesting preclinical and clinical candidates. PMID:21558000

  16. The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer.

    PubMed

    Tew, Kenneth D; Manevich, Yefim; Grek, Christina; Xiong, Ying; Uys, Joachim; Townsend, Danyelle M

    2011-07-15

    Glutathione S-transferase P is abundantly expressed in some mammalian tissues, particularly those associated with malignancies. While the enzyme can catalyze thioether bond formation between some electrophilic chemicals and GSH, novel nondetoxification functions are now ascribed to it. This review summarizes recent material that implicates GSTP in mediating S-glutathionylation of specific clusters of target proteins and in reactions that define a negative regulatory role in some kinase pathways through ligand or protein:protein interactions. It is becoming apparent that GSTP participates in the maintenance of cellular redox homeostasis through a number of convergent and divergent mechanisms. Moreover, drug platforms that have GSTP as a target have produced some interesting preclinical and clinical candidates. PMID:21558000

  17. Molecular and clonal analysis of in vivo hprt (hypoxanthine-guanine phosphoribosyl-transferase) mutations in human cells

    SciTech Connect

    Albertini, R.J.; O'Neill, J.P.; Nicklas, J.A.; Allegretta, M. . Genetics Lab.); Recio, L.; Skopek, T.R. )

    1989-08-08

    There is no longer doubt that gene mutations occur in vivo in human somatic cells, and that methods can be developed to detect, quantify and study them. Four assays are now available for such purpose; two detecting mutations that arise in bone marrow erythroid stem cells and two defining mutations that occur in T-lymphocytes. The red cell assays measure changes in mature red blood cells that involve either the blood group glycophorin-A locus or the hemoglobin loci; the lymphocyte assays score for genetic events at either the X-chromosomal hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus. We describe here our attempts in studying in vivo gene mutations in human T-lymphocytes. 35 refs., 3 figs., 3 tabs.

  18. Mimicking Insect Communication: Release and Detection of Pheromone, Biosynthesized by an Alcohol Acetyl Transferase Immobilized in a Microreactor

    PubMed Central

    Muñoz, Lourdes; Dimov, Nikolay; Carot-Sans, Gerard; Bula, Wojciech P.; Guerrero, Angel; Gardeniers, Han J. G. E.

    2012-01-01

    Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis, is achieved in a novel microfluidic system designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an “artificial gland”, i.e., a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behavior as 3 virgin females in most of the parameters considered. PMID:23155372

  19. Loss-of-function mutations in a glutathione S-transferase suppress the prune-Killer of prune lethal interaction.

    PubMed

    Provost, Elayne; Hersperger, Grafton; Timmons, Lisa; Ho, Wen Qi; Hersperger, Evelyn; Alcazar, Rosa; Shearn, Allen

    2006-01-01

    The prune gene of Drosophila melanogaster is predicted to encode a phosphodiesterase. Null alleles of prune are viable but cause an eye-color phenotype. The abnormal wing discs gene encodes a nucleoside diphosphate kinase. Killer of prune is a missense mutation in the abnormal wing discs gene. Although it has no phenotype by itself even when homozygous, Killer of prune when heterozygous causes lethality in the absence of prune gene function. A screen for suppressors of transgenic Killer of prune led to the recovery of three mutations, all of which are in the same gene. As heterozygotes these mutations are dominant suppressors of the prune-Killer of prune lethal interaction; as homozygotes these mutations cause early larval lethality and the absence of imaginal discs. These alleles are loss-of-function mutations in CG10065, a gene that is predicted to encode a protein with several zinc finger domains and glutathione S-transferase activity. PMID:16143620

  20. Transgenic Arabidopsis Plants Expressing Tomato Glutathione S-Transferase Showed Enhanced Resistance to Salt and Drought Stress.

    PubMed

    Xu, Jing; Xing, Xiao-Juan; Tian, Yong-Sheng; Peng, Ri-He; Xue, Yong; Zhao, Wei; Yao, Quan-Hong

    2015-01-01

    Although glutathione S-transferases (GST, EC 2.5.1.18) are involved in response to abiotic stress, limited information is available regarding gene function in tomato. In this study, a GST gene from tomato, designated LeGSTU2, was cloned and functionally characterized. Expression profile analysis results showed that it was expressed in roots and flowers, and the transcription was induced by salt, osmotic, and heat stress. The gene was then introduced to Arabidopsis by Agrobacterium tumefaciens-mediated transformation. Transgenic Arabidopsis plants were normal in terms of growth and maturity compared with wild-type plants. Transgenic plants also showed an enhanced resistance to salt and osmotic stress induced by NaCl and mannitol. The increased tolerance of transgenic plants was correlated with the changes in proline, malondialdehyde and antioxidative emzymes activities. Our results indicated that the gene from tomato plays a positive role in improving tolerance to salinity and drought stresses in Arabidopsis. PMID:26327625

  1. Genetic polymorphisms in glutathione-S-transferases are associated with anxiety and mood disorders in nicotine dependence

    PubMed Central

    Pizzo de Castro, Márcia Regina; Ehara Watanabe, Maria Angelica; Losi Guembarovski, Roberta; Odebrecht Vargas, Heber; Vissoci Reiche, Edna Maria; Kaminami Morimoto, Helena; Dodd, Seetal; Berk, Michael

    2014-01-01

    Background Nicotine dependence is associated with an increased risk of mood and anxiety disorders and suicide. The primary hypothesis of this study was to identify whether the polymorphisms of two glutathione-S-transferase enzymes (GSTM1 and GSTT1 genes) predict an increased risk of mood and anxiety disorders in smokers with nicotine dependence. Materials and methods Smokers were recruited at the Centre of Treatment for Smokers. The instruments were a sociodemographic questionnaire, Fagerström Test for Nicotine Dependence, diagnoses of mood disorder and nicotine dependence according to DSM-IV (SCID-IV), and the Alcohol, Smoking and Substance Involvement Screening Test. Anxiety disorder was assessed based on the treatment report. Laboratory assessment included glutathione-S-transferases M1 (GSTM1) and T1 (GSTT1), which were detected by a multiplex-PCR protocol. Results Compared with individuals who had both GSTM1 and GSTT1 genes, a higher frequency of at least one deletion of the GSTM1 and GSTT1 genes was identified in anxious smokers [odds ratio (OR)=2.21, 95% confidence interval (CI)=1.05–4.65, P=0.034], but there was no association with bipolar and unipolar depression (P=0.943). Compared with nonanxious smokers, anxious smokers had a greater risk for mood disorders (OR=4.67; 95% CI=2.24–9.92, P<0.001), lung disease (OR=6.78, 95% CI=1.95–23.58, P<0.003), and suicide attempts (OR=17.01, 95% CI=2.23–129.91, P<0.006). Conclusion This study suggests that at least one deletion of the GSTM1 and GSTT1 genes represents a risk factor for anxious smokers. These two genes may modify the capacity for the detoxification potential against oxidative stress. PMID:24637631

  2. Human monomethylarsonic acid (MMA(V)) reductase is a member of the glutathione-S-transferase superfamily.

    PubMed

    Zakharyan, R A; Sampayo-Reyes, A; Healy, S M; Tsaprailis, G; Board, P G; Liebler, D C; Aposhian, H V

    2001-08-01

    The drinking of water containing large amounts of inorganic arsenic is a worldwide major public health problem because of arsenic carcinogenicity. Yet an understanding of the specific mechanism(s) of inorganic arsenic toxicity has been elusive. We have now partially purified the rate-limiting enzyme of inorganic arsenic metabolism, human liver MMA(V) reductase, using ion exchange, molecular exclusion, and hydroxyapatite chromatography. When SDS-beta-mercaptoethanol-PAGE was performed on the most purified fraction, seven protein bands were obtained. Each band was excised from the gel, sequenced by LC-MS/MS and identified according to the SWISS-PROT and TrEMBL Protein Sequence databases. Human liver MMA(V) reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered human glutathione-S-transferase Omega class hGSTO 1-1. Recombinant human GSTO1-1 had MMA(V) reductase activity with K(m) and V(max) values comparable to those of human liver MMA(V) reductase. The partially purified human liver MMA(V) reductase had glutathione S-transferase (GST) activity. MMA(V) reductase activity was competitively inhibited by the GST substrate, 1-chloro 2,4-dinitrobenzene and also by the GST inhibitor, deoxycholate. Western blot analysis of the most purified human liver MMA(V) reductase showed one band when probed with hGSTO1-1 antiserum. We propose that MMA(V) reductase and hGSTO 1-1 are identical proteins. PMID:11511179

  3. Glutathione S-transferase M1 polymorphism and esophageal cancer risk: An updated meta-analysis based on 37 studies

    PubMed Central

    Lu, Quan-Jun; Bo, Ya-Cong; Zhao, Yan; Zhao, Er-Jiang; Sapa, Wolde Bekalo; Yao, Ming-Jie; Duan, Dan-Dan; Zhu, Yi-Wei; Lu, Wei-Quan; Yuan, Ling

    2016-01-01

    AIM: To evaluate the relationship between glutathione S-transferase M1 (GSTM1) polymorphism and susceptibility to esophageal cancer (EC). METHODS: A comprehensive search of the United States National Library of Medicine PubMed database and the Elsevier, Springer, and China National Knowledge Infrastructure databases for all relevant studies was conducted using combinations of the following terms: “glutathione S-transferase M1”, “GSTM1”, “polymorphism”, and “EC” (until November 1, 2014). The statistical analysis was performed using the SAS software (v.9.1.3; SAS Institute, Cary, NC, United States) and the Review Manager software (v.5.0; Oxford, England); crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the association between the GSTM1 null genotype and the risk of EC. RESULTS: A total of 37 studies involving 2236 EC cases and 3243 controls were included in this meta-analysis. We observed that the GSTM1 null genotype was a significant risk factor for EC in most populations (OR = 1.33, 95%CI: 1.12-1.57, Pheterogeneity < 0.000001, and I2 = 77.0%), particularly in the Asian population (OR = 1.53, 95%CI: 1.26-1.86, Pheterogeneity < 0.000001, and I2 = 77.0%), but not in the Caucasian population (OR = 1.02, 95%CI: 0.87-1.19, Pheterogeneity = 0.97, and I2 = 0%). CONCLUSION: The GSTM1 null polymorphism may be associated with an increased risk for EC in Asian but not Caucasian populations. PMID:26855551

  4. Effects of gestational and overt diabetes on human placental cytochromes P450 and glutathione S-transferase.

    PubMed

    McRobie, D J; Glover, D D; Tracy, T S

    1998-04-01

    The placenta possesses the ability to metabolize a number of xenobiotics and endogenous compounds by processes similar to those seen in the liver. Animal and in vivo studies have observed that the presence of diabetes alters the expression of hepatic metabolizing enzymes (cytochrome P450 and glutathione S-transferase); however, it is unknown whether similar alterations occur in the human placenta. To evaluate whether diabetes has any effect of placental xenobiotic metabolizing activity, the catalytic activities of 7-ethoxyresorufin O-deethylation (EROD, CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2, 4-dinitrobenzene (CDNB) conjugation with glutathione (glutathione S-transferase, GST) from placentas of diet (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared with matched controls. EROD activity (CYP1A1) ranged from 0.29 to 2.67 pmol/min/mg protein. However, no differences were observed among overt or gestational diabetics and their respective matched controls. CDNB conjugation (GST) ranged from 0.275 to 1.65 units/min/mg protein. In contrast to that observed with CYP1A1, a small but statistically significant reduction in GST activity was noted in overt diabetics as compared with their matched controls and gestational diabetics. CYP2E1, 2D6, and 3A4 enzymatic activities were not detected in human placental tissue. GST protein was detectable in all tissues studied, but no CYP protein could be detected in any of the tissues. Thus, it seems that pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in the exposure of the fetus to harmful electrophiles. However, the full clinical significance of this finding remains to be elucidated. PMID:9531526

  5. Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase.

    PubMed

    Toyota, Cory G; Berthold, Catrine L; Gruez, Arnaud; Jónsson, Stefán; Lindqvist, Ylva; Cambillau, Christian; Richards, Nigel G J

    2008-04-01

    The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions. PMID:18245280

  6. Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs in Arabidopsis.

    PubMed

    Wang, Xiaoyan; Zhang, Shuxin; Dou, Yongchao; Zhang, Chi; Chen, Xuemei; Yu, Bin; Ren, Guodong

    2015-04-01

    All types of small RNAs in plants, piwi-interacting RNAs (piRNAs) in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3' terminal 2'-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3' uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an) enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1) is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3'-to-5' trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3' uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3' end modification and stability control. PMID:25928341

  7. Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

    PubMed

    Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

    2014-06-01

    Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. PMID:24654730

  8. Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5

    PubMed Central

    Summers, Ryan M.; Seffernick, Jennifer L.; Quandt, Erik M.; Yu, Chi Li; Barrick, Jeffrey E.

    2013-01-01

    Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N1- and N3-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N7-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners. PMID:23813729

  9. p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

    PubMed Central

    Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

    2014-01-01

    Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

  10. The silkworm glutathione S-transferase gene noppera-bo is required for ecdysteroid biosynthesis and larval development.

    PubMed

    Enya, Sora; Daimon, Takaaki; Igarashi, Fumihiko; Kataoka, Hiroshi; Uchibori, Miwa; Sezutsu, Hideki; Shinoda, Tetsuro; Niwa, Ryusuke

    2015-06-01

    Insect molting and metamorphosis are tightly controlled by ecdysteroids, which are important steroid hormones that are synthesized from dietary sterols in the prothoracic gland. One of the ecdysteroidogenic genes in the fruit fly Drosophila melanogaster is noppera-bo (nobo), also known as GSTe14, which encodes a member of the epsilon class of glutathione S-transferases. In D. melanogaster, nobo plays a crucial role in utilizing cholesterol via regulating its transport and/or metabolism in the prothoracic gland. However, it is still not known whether the orthologs of nobo from other insects are also involved in ecdysteroid biosynthesis via cholesterol transport and/or metabolism in the prothoracic gland. Here we report genetic evidence showing that the silkworm Bombyx mori ortholog of nobo (nobo-Bm; GSTe7) is essential for silkworm development. nobo-Bm is predominantly expressed in the prothoracic gland. To assess the functional importance of nobo-Bm, we generated a B. mori genetic mutant of nobo-Bm using TALEN-mediated genome editing. We show that loss of nobo-Bm function causes larval arrest and a glossy cuticle phenotype, which are rescued by the application of 20-hydroxyecdysone. Moreover, the prothoracic gland cells isolated from the nobo-Bm mutant exhibit an abnormal accumulation of 7-dehydrocholesterol, a cholesterol metabolite. These results suggest that the nobo family of glutathione S-transferases is essential for development and for the regulation of sterol utilization in the prothoracic gland in not only the Diptera but also the Lepidoptera. On the other hand, loss of nobo function mutants of D. melanogaster and B. mori abnormally accumulates different sterols, implying that the sterol utilization in the PG is somewhat different between these two insect species. PMID:25881968

  11. GAMMA-GLUTAMYL TRANSFERASE (GGT) ACTIVITY AND BIOCHEMICAL CHARACTERIZATION OF RAT VISCERAL YOLK-SAC DURING GESTATION WITH OR WITHOUT TRYPAN BLUE EXPOSURE

    EPA Science Inventory

    Yolk-sacs from untreated Sprague Dawley rat conceptuses were removed on days 9-18 of gestation and examined for gamma-glutamyl transferase (GGT), alkaline phosphatase (AP), lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT) activities. All enzyme activities w...

  12. INDUCTION OF DNA-PROTEIN CROSSLINKS BY THE METABOLISM OF DICHLOROMETHANE IN V79 CELL LINES TRANSFECTED WITH THE MURINE GLUTATHIONE-S-TRANSFERASE THETA 1 GENE

    EPA Science Inventory

    Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and...

  13. Differential transcription of cytochrome P450s and glutathione S transferases in DDT-susceptible and resistant Drosophila melanogaster strains in response to DDT and oxidative stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been...

  14. Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzym...

  15. Cold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathione transferase isoenzyme GSTZ2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GSTZs (zeta class glutathione transferases) belong to a highly conserved subfamily of soluble GSTs found in species ranging from fungi and plants to animals. GSTZ is identical to MAAI (maleylacetoacetate isomerase), which functions in tyrosine catabolism by catalyzing the isomerization of MAA (maley...

  16. Characterization of Alcohol Acyl Transferase and 1-Aminocyclopropane-1-Carboxylate Synthase Gene Expression and Volatile Compound Emission during Apple Fruit Development and Ripening

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alcohol acyl transferase (AAT) catalyzes the last step of volatile ester biosynthesis, and in this study, expression of four apple AAT genes was investigated in the peel of two apple cultivars with relatively high (‘Golden Delicious’) or low (‘Granny Smith’) volatile ester production. All four AAT ...

  17. The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat.

    PubMed Central

    Shrader, T E; Tobias, J W; Varshavsky, A

    1993-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule operate in bacteria, fungi, and mammals. We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway. L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine. The aat gene maps to the 19-min region of the E. coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases. In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine. However, the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the beta-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp. Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed. The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins. PMID:8331068

  18. Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Steindorf, Astrid; Alber, Birgit E; Fuchs, Georg

    2006-04-01

    The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle. PMID:16547052

  19. Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family

    PubMed Central

    Schürmann, Marc; Hirsch, Beatrice; Wübbeler, Jan Hendrik; Stöveken, Nadine

    2013-01-01

    The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, ActTBEA6 (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3′-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated ActTBEA6 to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. ActTBEA6 was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent Vmax of 44.6 μmol min−1 mg−1 for succinyl-CoA, which corresponds to a turnover number of 36.0 s−1 per subunit of ActTBEA6. For 3SP, the apparent Vmax was determined as 46.8 μmol min−1 mg−1, which corresponds to a turnover number of 37.7 s−1 per subunit of ActTBEA6. The apparent Km values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus Δact mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon

  20. Inhibition of O-Linked N-Acetylglucosamine Transferase Reduces Replication of Herpes Simplex Virus and Human Cytomegalovirus

    PubMed Central

    Angelova, Magdalena; Ortiz-Meoz, Rodrigo F.; Walker, Suzanne

    2015-01-01

    ABSTRACT O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies nuclear and cytoplasmic proteins via O-linked addition of a single N-acetylglucosamine (GlcNAc) moiety. Among the many targets of OGT is host cell factor 1 (HCF-1), a transcriptional regulator that is required for transactivation of the immediate-early genes of herpes simplex virus (HSV). HCF-1 is synthesized as a large precursor that is proteolytically cleaved by OGT, which may regulate its biological function. In this study, we tested whether inhibition of the enzymatic activity of OGT with a small molecule inhibitor, OSMI-1, affects initiation of HSV immediate-early gene expression and viral replication. We found that inhibiting OGT's enzymatic activity significantly decreased HSV replication. The major effect of the inhibitor occurred late in the viral replication cycle, when it reduced the levels of late proteins and inhibited capsid formation. However, depleting OGT levels with small interfering RNA (siRNA) reduced the expression of HSV immediate-early genes, in addition to reducing viral yields. In this study, we identified OGT as a novel cellular factor involved in HSV replication. Our results obtained using a small molecule inhibitor and siRNA depletion suggest that OGT's glycosylation and scaffolding functions play distinct roles in the replication cycle of HSV. IMPORTANCE Antiviral agents can target viral or host gene products essential for viral replication. O-GlcNAc transferase (OGT) is an important cellular enzyme that catalyzes the posttranslational addition of GlcNAc sugar residues to hundreds of nuclear and cytoplasmic proteins, and this modification regulates their activity and function. Some of the known OGT targets are cellular proteins that are critical for the expression of herpes simplex virus (HSV) genes, suggesting a role for OGT in the replication cycle of HSV. In this study, we found that OGT is required for

  1. Thiopurine metabolites variations during co-treatment with aminosalicylates for inflammatory bowel disease: Effect of N-acetyl transferase polymorphisms

    PubMed Central

    Stocco, Gabriele; Cuzzoni, Eva; De Iudicibus, Sara; Favretto, Diego; Malusà, Noelia; Martelossi, Stefano; Pozzi, Elena; Lionetti, Paolo; Ventura, Alessandro; Decorti, Giuliana

    2015-01-01

    AIM: To evaluate variation of the concentration of thiopurine metabolites after 5-aminosalicylate (5-ASA) interruption and the role of genetic polymorphisms of N-acetyl transferase (NAT) 1 and 2. METHODS: Concentrations of thioguanine nucleotides (TGN) and methymercaptopurine nucleotides (MMPN), metabolites of thiopurines, were measured by high performance liquid chromatography in 12 young patients (3 females and 9 males, median age 16 years) with inflammatory bowel disease (6 Crohn’s disease and 6 ulcerative colitis) treated with thiopurines (7 mercaptopurine and 5 azathioprine) and 5-ASA. Blood samples were collected one month before and one month after the interruption of 5-ASA. DNA was extracted and genotyping of NAT1, NAT2, inosine triphosphate pyrophosphatase (ITPA) and thiopurine methyl transferase (TPMT) genes was performed using PCR assays. RESULTS: Median TGN concentration before 5-ASA interruption was 270 pmol/8 x 108 erythrocytes (range: 145-750); after the interruption of the aminosalicylate, a 35% reduction in TGN mean concentrations (absolute mean reduction 109 pmol/8 × 108 erythrocytes) was observed (median 221 pmol/8 × 108 erythrocytes, range: 96-427, P value linear mixed effects model 0.0011). Demographic and clinical covariates were not related to thiopurine metabolites concentrations. All patients were wild-type for the most relevant ITPA and TPMT variants. For NAT1 genotyping, 7 subjects presented an allele combination corresponding to fast enzymatic activity and 5 to slow activity. NAT1 genotypes corresponding to fast enzymatic activity were associated with reduced TGN concentration (P value linear mixed effects model 0.033), putatively because of increased 5-ASA inactivation and consequent reduced inhibition of thiopurine metabolism. The effect of NAT1 status on TGN seems to be persistent even after one month since the interruption of the aminosalicylate. No effect of NAT1 genotypes was shown on MMPN concentrations. NAT2 genotyping

  2. Proteome Biomarkers in Xylem Reveal Pierce’s Disease Tolerance in Grape

    PubMed Central

    Katam, Ramesh; Chibanguza, Kundai; Latinwo, Lekan M; Smith, Danyel

    2016-01-01

    Pierce’s disease (PD) is a significant threat to grape cultivation and industry. The disease caused by bacterium Xylella fastidiosa clogs xylem vessels resulting in wilting of the plant. PD-tolerant grape genotypes are believed to produce certain novel components in xylem tissue that help them to combat invading pathogens. Research has been aimed at characterizing the uniquely expressed xylem proteins by PD-tolerant genotypes. The objectives were to i) compare and characterize Vitis xylem proteins differentially expressed in PD-tolerant and PD-susceptible cultivars and, ii) identify xylem proteins uniquely expressed in PD-tolerant genotypes. A high throughput two-dimensional gel electrophoresis of xylem proteins from three Vitis species identified more than 200 proteins with pls 3.0 to 9.0 and molecular weights of 20 to 75 kDa. The differentially expressed proteins were then excised and analyzed with MALDI/TOF mass spectrometer. The mass spectra were collected and protein identification was performed against the Viridiplantae database using Matrix Science algorithm. Proteins were mapped to the universal protein resource to study gene ontology. Comparative analysis of the xylem proteome of three species indicated the highest number of proteins in muscadine grape, followed by Florida hybrid bunch and bunch grape. These proteins were all associated with disease resistance, energy metabolism, protein processing and degradation, biosynthesis, stress related functions, cell wall biogenesis, signal transduction, and ROS detoxification. Furthermore, β-1, 3-glucanase, 10-deacetyl baccatin III-10-O-acetyl transferase-like, COP9, and aspartyl protease nepenthesin precursor proteins were found to be uniquely expressed in PD-tolerant muscadine grape, while they are absent in PD-susceptible bunch grape. Data suggests that muscadine and Florida hybrid bunch grapes express novel proteins in xylem to overcome pathogen attack while bunch grape lacks this capability, making them

  3. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  4. Kinetic analysis of the intracellular conjugation of monochlorobimane by IC-21 murine macrophage glutathione-S-transferase.

    PubMed

    Young, P R; ConnorsWhite, A L; Dzido, G A

    1994-12-15

    Monochlorobimane (MCB) reacts with glutathione (GSH) in a reaction catalyzed by the glutathione-S-transferase (GST) isozymes. The diffusion of MCB through cell membranes is rapid and the fluorescence conjugates are relatively insensitive to quenching and to pH effects, and are expelled slowly from the cell, allowing the rate of fluorescence increase to be used to probe the dynamics of the intracellular reaction. Using low-light microscopic cytometry to monitor the initial rates of fluorescence increase for the GST-catalyzed reaction within IC-21 macrophages yields Vmax = 8.4 x 10(-16) mol s-1 cell-1 and KMCBm = 65 microM. Combining these data with an integrated Michaelis analysis of the reaction course yields KIP approximately 1.5 x 10(-5) M, and KmGSH approximately 3.0 x 10(-4) M (at [MCB] = 50 microM). The values of Vmax and KMCBm for the cell-free (extracellular) GST-catalyzed conjugation reaction are 1.2 x 10(-18) mol s-1 cell-1 and 3.1 microM, respectively. The values of Vmax for the intra- and extracellular conjugation reactions differ by 700-fold, suggesting the presence of an intracellular activator for this enzyme system. PMID:7803478

  5. Molecular Cloning, Characterization and Positively Selected Sites of the Glutathione S-Transferase Family from Locusta migratoria

    PubMed Central

    Zhang, Min; Qin, Guohua; Li, Daqi; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2014-01-01

    Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes—sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses—insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily. PMID:25486043

  6. Organophosphate pesticides increase the expression of alpha glutathione S-transferase in HepG2 cells.

    PubMed

    Medina-Díaz, I M; Rubio-Ortíz, M; Martínez-Guzmán, M C; Dávalos-Ibarra, R L; Rojas-García, A E; Robledo-Marenco, M L; Barrón-Vivanco, B S; Girón-Pérez, M I; Elizondo, G

    2011-12-01

    Chlorpyrifos and methyl parathion are among the most widely used insecticides in the world. Human populations are constantly exposed to low doses of both due to their extensive use and presence in food and drinking water. Glutathione S-transferase (GST) catalyzes the conjugation of glutathione on electrophilic substrates and is an important line of defense in the protection of cellular components from reactive species. GST alpha1 (GSTA1) is the predominant isoform of GST expressed in the human liver; thus, determining the effect of insecticides on GSTA1 transcription is very important. In the present study, we analyzed the effects of methyl parathion and chlorpyrifos on GSTA1 gene expression in HepG2 cells using real time PCR, and activity and immunoreactive protein assays. The results demonstrated that exposure to methyl parathion and chlorpyrifos increased the level of GSTA1 mRNA, GSTA1 immunoreactive protein and GST activity relative to a control. These results demonstrated that these insecticides can increase the expression of GSTA1. In conclusion, HepG2 cell cultures treated with methyl parathion and chlorpyrifos could be a useful model for studying the function of GSTA1 and its role in the metabolism of xenobiotics in the liver. PMID:21907274

  7. Volatile Gas Production by Methyl Halide Transferase: An In Situ Reporter Of Microbial Gene Expression In Soil.

    PubMed

    Cheng, Hsiao-Ying; Masiello, Caroline A; Bennett, George N; Silberg, Jonathan J

    2016-08-16

    Traditional visual reporters of gene expression have only very limited use in soils because their outputs are challenging to detect through the soil matrix. This severely restricts our ability to study time-dependent microbial gene expression in one of the Earth's largest, most complex habitats. Here we describe an approach to report on dynamic gene expression within a microbial population in a soil under natural water levels (at and below water holding capacity) via production of methyl halides using a methyl halide transferase. As a proof-of-concept application, we couple the expression of this gas reporter to the conjugative transfer of a bacterial plasmid in a soil matrix and show that gas released from the matrix displays a strong correlation with the number of transconjugant bacteria that formed. Gas reporting of gene expression will make possible dynamic studies of natural and engineered microbes within many hard-to-image environmental matrices (soils, sediments, sludge, and biomass) at sample scales exceeding those used for traditional visual reporting. PMID:27415416

  8. The Plasmodium palmitoyl-S-acyl-transferase DHHC2 is essential for ookinete morphogenesis and malaria transmission

    PubMed Central

    Santos, Jorge M.; Kehrer, Jessica; Franke-Fayard, Blandine; Frischknecht, Friedrich; Janse, Chris J.; Mair, Gunnar R.

    2015-01-01

    The post-translational addition of C-16 long chain fatty acids to protein cysteine residues is catalysed by palmitoyl-S-acyl-transferases (PAT) and affects the affinity of a modified protein for membranes and therefore its subcellular localisation. In apicomplexan parasites this reversible protein modification regulates numerous biological processes and specifically affects cell motility, and invasion of host cells by Plasmodium falciparum merozoites and Toxoplasma gondii tachyzoites. Using inhibitor studies we show here that palmitoylation is key to transformation of zygotes into ookinetes during initial mosquito infection with P. berghei. We identify DHHC2 as a unique PAT mediating ookinete formation and morphogenesis. Essential for life cycle progression in asexual blood stage parasites and thus refractory to gene deletion analyses, we used promoter swap (ps) methodology to maintain dhhc2 expression in asexual blood stages but down regulate expression in sexual stage parasites and during post-fertilization development of the zygote. The ps mutant showed normal gamete formation, fertilisation and DNA replication to tetraploid cells, but was characterised by a complete block in post-fertilisation development and ookinete formation. Our report highlights the crucial nature of the DHHC2 palmitoyl-S-acyltransferase for transmission of the malaria parasite to the mosquito vector through its essential role for ookinete morphogenesis. PMID:26526684

  9. Glutathione S-transferase activity influences busulfan pharmacokinetics in patients with beta thalassemia major undergoing bone marrow transplantation.

    PubMed

    Poonkuzhali, B; Chandy, M; Srivastava, A; Dennison, D; Krishnamoorthy, R

    2001-03-01

    Busulfan, at a dose of 16 mg/kg, is widely used in combination with cyclophosphamide as a conditioning regimen for patients undergoing bone marrow transplantation. Wide interindividual variation in busulfan kinetics and rapid clearance of the drug have been reported, especially in children. Some of the factors contributing to interpatient variability have been identified. They include circadian rhythms, age, disease, drug interaction, changes in hepatic function, and busulfan bioavailability. In this study, we demonstrate that hepatic glutathione S-transferase (GST) activity correlates negatively with busulfan maximum and minimum concentrations (Pearson's correlation r = -0.74 and -0.77, respectively) and positively with busulfan clearance (Pearson's correlation r = 0.728) in children with thalassemia major in the age range of 2 to 15 years. We also found that plasma alpha GST levels were 5 to 10 times higher in patients with thalassemia than in normal controls and age-matched leukemic patients, either reflecting extensive liver damage, elevated expression of the enzyme, or both in thalassemic patients. Plasma alpha GST concentrations showed a similar correlation with busulfan kinetic parameters to that observed for hepatic GST. The status of hepatic GST activity accounts, at least in part, for the observed interindividual variation in busulfan kinetics, while the observed association with plasma alpha GST is difficult to explain at present. PMID:11181493

  10. Pi class glutathione S-transferase genes are regulated by Nrf 2 through an evolutionarily conserved regulatory element in zebrafish

    PubMed Central

    Suzuki, Takafumi; Takagi, Yaeko; Osanai, Hitoshi; Li, Li; Takeuchi, Miki; Katoh, Yasutake; Kobayashi, Makoto; Yamamoto, Masayuki

    2005-01-01

    Pi class GSTs (glutathione S-transferases) are a member of the vertebrate GST family of proteins that catalyse the conjugation of GSH to electrophilic compounds. The expression of Pi class GST genes can be induced by exposure to electrophiles. We demonstrated previously that the transcription factor Nrf 2 (NF-E2 p45-related factor 2) mediates this induction, not only in mammals, but also in fish. In the present study, we have isolated the genomic region of zebrafish containing the genes gstp1 and gstp2. The regulatory regions of zebrafish gstp1 and gstp2 have been examined by GFP (green fluorescent protein)-reporter gene analyses using microinjection into zebrafish embryos. Deletion and point-mutation analyses of the gstp1 promoter showed that an ARE (antioxidant-responsive element)-like sequence is located 50 bp upstream of the transcription initiation site which is essential for Nrf 2 transactivation. Using EMSA (electrophoretic mobility-shift assay) analysis we showed that zebrafish Nrf 2–MafK heterodimer specifically bound to this sequence. All the vertebrate Pi class GST genes harbour a similar ARE-like sequence in their promoter regions. We propose that this sequence is a conserved target site for Nrf 2 in the Pi class GST genes. PMID:15654768

  11. Circadian regulation of permethrin susceptibility by glutathione S-transferase (BgGSTD1) in the German cockroach (Blattella germanica).

    PubMed

    Lin, Yu-Hsien; Lee, Chi-Mei; Huang, Jia-Hsin; Lee, How-Jing

    2014-06-01

    The daily susceptibility rhythm to permethrin and the expression level of the delta class glutathione S-transferase (BgGSTD1) gene were investigated in Blattella germanica. Male cockroaches were exposed to the same concentration of permethrin at different times in a light-dark cycle, and results showed that the highest resistance occurred at night. Furthermore, the circadian rhythmicity of permethrin susceptibility was demonstrated by the highest resistance at subjective night under constant darkness. The mRNA level of the BgGSTD1 gene in the fat body of B. germanica peaked early in the day or subjective day under light-dark or constant dark conditions, whereas enzyme activity of cytosolic GSTs did not reflect the rhythmic pattern as well as BgGSTD1 expression. RNA interference (RNAi) was employed to study the function of BgGSTD1 in the circadian rhythm of permethrin susceptibility in B. germanica. Both BgGSTD1 mRNA level and cytosolic GSTs activity were significantly decreased by dsGSTD1 injection. In addition, survival of B. germanica with silenced BgGSTD1 was significantly decreased at night but not in the day when the cockroaches were exposed to permethrin. Total cytosolic GSTs activity demonstrated that is not the only gene involved in the circadian regulation of the permethrin resistance, although it is one of the major regulators of permethrin resistance. PMID:24819204

  12. Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif.

    PubMed Central

    Yalovsky, S.; Loraine, A. E.; Gruissem, W.

    1996-01-01

    Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity. PMID:12226265

  13. Control of larval and egg development in Aedes aegypti with RNA interference against juvenile hormone acid methyl transferase.

    PubMed

    Van Ekert, Evelien; Powell, Charles A; Shatters, Robert G; Borovsky, Dov

    2014-11-01

    RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including mosquitoes and many other insects. Little has been done, however, to harness this approach in order to control adult and larval mosquitoes. Juvenile hormone (JH) plays a pivotal role in the control of reproduction in adults and metamorphism in larval mosquitoes. This report describes an approach to control Aedes aegypti using RNAi against JH acid methyl transferase (AeaJHAMT), the ultimate enzyme in the biosynthetic pathway of JH III that converts JH acid III (JHA III) into JH III. In female A. aegypti that were injected or fed jmtA dsRNA targeting the AeaJHAMT gene (jmtA) transcript, egg development was inhibited in 50% of the treated females. In mosquito larvae that were fed transgenic Pichia pastoris cells expressing long hair pin (LHP) RNA, adult eclosion was delayed by 3 weeks causing high mortality. Northern blot analyses and qPCR studies show that jmtA dsRNA causes inhibition of jmtA transcript in adults and larvae, which is consistent with the observed inhibition of egg maturation and larval development. Taken together, these results suggest that jmtA LHP RNA expressed in heat inactivated genetically modified P. pastoris cells could be used to control mosquito populations in the marsh. PMID:25111689

  14. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    PubMed Central

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  15. Nitration of succinyl-CoA:3-oxoacid CoA-transferase in rats after endotoxin administration

    PubMed Central

    Marcondes, Sisi; Turko, Illarion V.; Murad, Ferid

    2001-01-01

    The tyrosine nitration of proteins has been observed in diverse inflammatory conditions and has been linked to the presence of reactive nitrogen species. From many in vitro experiments, it is apparent that tyrosine nitration may alter the function of proteins. A limited number of experiments under in vivo conditions also demonstrate that protein nitration is associated with altered cellular processes. To understand the association of protein nitration with the pathogenic mechanism of the disease, it is essential to identify specific protein targets of nitration with in vivo or intact tissue models. Using anti-nitrotyrosine antibodies, we demonstrated the accumulation of nitrotyrosine in a 52-kDa protein in rat kidney after lipopolysaccharide treatment. The 52-kDa protein was purified and identified with partial sequence as succinyl-CoA:3-oxoacid CoA-transferase (SCOT; EC 2.8.3.5). Western blot analysis revealed that the nitration of this mitochondrial enzyme increased in the kidneys and hearts of lipopolysaccharide-treated rats, whereas its catalytic activity decreased. These data suggest that tyrosine nitration may be a mechanism for the inhibition of SCOT activity in inflammatory conditions. SCOT is a key enzyme for ketone body utilization. Thus, tyrosine nitration of the enzyme with sepsis or inflammation may explain the altered metabolism of ketone bodies present in these disorders. PMID:11416199

  16. Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase.

    PubMed

    Jaffee, Marcie B; Imperiali, Barbara

    2013-06-01

    Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of Escherichia coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein. PMID:23583934

  17. Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase

    PubMed Central

    Jaffee, Marcie B.; Imperiali, Barbara

    2013-01-01

    Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of E. coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein. PMID:23583934

  18. Identification of Class-mu glutathione transferase genes GSTMI-GSTM5 on human chromosome lpl3

    SciTech Connect

    Pearson, W.R.; Vorachek, W.R.; Xu, Shi-jie ); Berger, R.; Hart, I.; Vannais, D.; Patterson, D. )

    1993-07-01

    The GSTM1, GSTM2, GSTM3, GSTM4, and GSTM5 glutathione transferase genes have been mapped to human chromosome 1 by using locus-specific PCR primer pairs spanning exon 6, intron 6, and exon 7, as probes on DNA from human/hamster somatic cell hybrids. For GSTM1, the assignment was confirmed by Southern blot hybridization to a pair of 12.5/2.4-kb HindlIl fragments. The GSTM1-specific primer pairs can be used to identify individuals carrying non-null GSTM1 alleles. The organization of these five genes was confirmed by the isolation of a yeast artificial chromosome clone (GSTM-YAC2) that contains all five genes. With this clone, the location of the GSTM1-GSTM5 gene cluster on chromosome 1 was confirmed by fluorescence in situ hybridization. Both regional assignment using the fractional length method and examination of probe signal with reference to R-banded chromosomes induced by BrdU places the gene cluster in or near the 1p13.3 region. The close physical proximity of the GSTM1 and GSTM2 loci, which share 99% nucleotide sequence identity over 460 nucleotides of 3'-untranslated mRNA, suggests that the GSTM1-null allele may result from unequal crossing-over. 49 refs., 8 figs., 1 tab.

  19. Effect of arsenic and chromium on the serum amino-transferases activity in Indian major carp, Labeo rohita.

    PubMed

    Vutukuru, Sesha Srinivas; Prabhath, N Arun; Raghavender, M; Yerramilli, Anjaneyulu

    2007-09-01

    Arsenic and hexavalent chromium toxicity results from their ability to interact with sulfahydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Alanine aminotransferase (ALT; E.C: 2.6.1.2) and Aspartate amino transferase (AST; EC 2.6.1.1) play a crucial role in transamination reactions and can be used as potential biomarkers to indicate hepatotoxicity and cellular damage. While histopathological studies in liver tissue require more time and expertise, simple and reliable biochemical analysis of ALT and AST can be used for a rapid assessment of tissue and cellular damage within 96 h. The main objective of this study was to determine the acute effects of arsenic and hexavalent chromium on the activity of ALT and AST in the Indian major carp, Labeo rohita for 24 h and 96 h. Significant increase in the activity of ALT (P < 0.01) from controls in arsenic exposed fish indicates serious hepatic damage and distress condition to the fish. However, no such significant changes were observed in chromium-exposed fish suggesting that arsenic is more toxic to the fish. These findings indicate that ALT and AST are candidate biomarkers for arsenic-induced hepatotoxicity in Labeo rohita. PMID:17911661

  20. Computational QM/MM Study of the Reaction Mechanism of Human Glutathione S-Transferase A3-3

    NASA Astrophysics Data System (ADS)

    Calvaresi, Matteo; Stenta, Marco; Altoè, Piero; Bottoni, Andrea; Garavelli, Marco; Spinelli, Domenico

    2007-12-01

    Human Glutathione S-Transferase A3-3(hGSTA3-3) is the most efficient human steroid double-bond isomerase enzyme. It catalyzes the double bond isomerization of Δ5-androstene-3,17-dione (Δ5-AD) and Δ5-pregnene-3,20-dione (Δ5-PD). The isomerization products are the precursors of the steroid hormones testosterone and progesterone. We have carried out a QM/MM study to elucidate some interesting aspects of the enzyme catalytic mechanism. In particular, we have analyzed either a concerted or a stepwise reaction path. Moreover, we have attempted to rationalize the electrostatic effects on the catalytic activity of the residues surrounding the active site. Specifically, we have performed a "finger print" analysis to determine the electrostatic contribution of each aminoacid residue to the global electrostatic term, thus ranking the effect of the various aminoacids in the course of the reaction. In this way, we have highlighted the most important terms affecting the stabilization-destabilization of the enzyme.

  1. Designer xanthone: an inhibitor scaffold for MDR-involved human glutathione transferase isoenzyme A1-1.

    PubMed

    Zoi, Ourania G; Thireou, Trias N; Rinotas, Vagelis E; Tsoungas, Petros G; Eliopoulos, Elias E; Douni, Eleni K; Labrou, Nikolaos E; Clonis, Yannis D

    2013-10-01

    Glutathione transferases (GSTs) are cell detoxifiers involved in multiple drug resistance (MDR), hampering the effectiveness of certain anticancer drugs. To our knowledge, this is the first report on well-defined synthetic xanthones as GST inhibitors. Screening 18 xanthones revealed three derivatives bearing a bromomethyl and a methyl group (7) or two bromomethyl groups (8) or an aldehyde group (17), with high inhibition potency (>85%), manifested by low IC(50) values (7: 1.59 ± 0.25 µM, 8: 5.30 ± 0.30 µM, and 17: 8.56 ± 0.14 µM) and a competitive modality of inhibition versus CDNB (Ki(7) = 0.76 ± 0.18 and Ki(17) = 1.69 ± 0.08 µM). Of them, derivative 17 readily inhibited hGSTA1-1 in colon cancer cell lysate (IC(50) = 10.54 ± 2.41 µM). Furthermore, all three derivatives were cytotoxic to Caco-2 intact cells, with 17 being the least cytotoxic (LC(50) = 151.3 ± 16.3 µM). The xanthone scaffold may be regarded as a pharmacophore for hGSTA1-1 and the three derivatives, especially 17, as potent precursors for the synthesis of new inhibitors and conjugate prodrugs for human GSTs. PMID:23749766

  2. Genetic variation in glutathione S-transferase genes and risk of nonfatal cerebral stroke in patients suffering from essential hypertension.

    PubMed

    Polonikov, Alexey; Vialykh, Ekaterina; Vasil'eva, Oksana; Bulgakova, Irina; Bushueva, Olga; Illig, Thomas; Solodilova, Maria

    2012-07-01

    Oxidative stress resulting from an increased amount of reactive oxygen species and an imbalance between oxidants and antioxidants has been implicated in pathogenesis of cerebral stroke. The purpose of this study was to investigate the relationship between common polymorphisms of glutathione S-transferase M1, T1, and P1 genes and risk of stroke in hypertensive individuals. A total of 667 unrelated Russian individuals with hypertension, including 306 hypertensives who suffered from cerebral stroke and 361 hypertensives who did not have cerebrovascular accidents, were recruited for the study. The deletion polymorphisms of GSTM1 and GSTT1 genes and polymorphism Ile105Val of the GSTP1 gene were genotyped by a multiplex polymerase chain reaction and restriction analyses, respectively. No differences in GSTM1 and GSTP1 genotype distributions between the cases and controls have been observed. The null GSTT1 genotype was found to be associated with increased risk of cerebral stroke after Bonferroni correction and adjusting for confounding variables such as gender, blood pressure, body mass index, and antihypertensive medication use (odds ratio 1.51 95 % CI 1.09-2.07, P = 0.01). The present study was the first to show the association of null genotype of the GSTT1 gene with increased risk of cerebral stroke. PMID:22528457

  3. Effect of Arsenic and Chromium on the Serum Amino-Transferases Activity in Indian Major Carp, Labeo rohita

    PubMed Central

    Vutukuru, Sesha Srinivas; Arun Prabhath, N.; Raghavender, M.; Yerramilli, Anjaneyulu

    2007-01-01

    Arsenic and hexavalent chromium toxicity results from their ability to interact with sulfahydryl groups of proteins and enzymes, and to substitute phosphorus in a variety of biochemical reactions. Alanine aminotransferase (ALT; E.C: 2.6.1.2) and Aspartate amino transferase (AST; EC 2.6.1.1) play a crucial role in transamination reactions and can be used as potential biomarkers to indicate hepatotoxicity and cellular damage. While histopathological studies in liver tissue require more time and expertise, simple and reliable biochemical analysis of ALT and AST can be used for a rapid assessment of tissue and cellular damage within 96 h. The main objective of this study was to determine the acute effects of arsenic and hexavalent chromium on the activity of ALT and AST in the Indian major carp, Labeo rohita for 24 h and 96 h. Significant increase in the activity of ALT (P < 0.01) from controls in arsenic exposed fish indicates serious hepatic damage and distress condition to the fish. However, no such significant changes were observed in chromium-exposed fish suggesting that arsenic is more toxic to the fish. These findings indicate that ALT and AST are candidate biomarkers for arsenic-induced hepatotoxicity in Labeo rohita. PMID:17911661

  4. Modification of the association between maternal smoke exposure and congenital heart defects by polymorphisms in glutathione S-transferase genes.

    PubMed

    Li, Xiaohong; Liu, Zhen; Deng, Ying; Li, Shengli; Mu, Dezhi; Tian, Xiaoxian; Lin, Yuan; Yang, Jiaxiang; Li, Jun; Li, Nana; Wang, Yanping; Chen, Xinlin; Deng, Kui; Zhu, Jun

    2015-01-01

    Congenital heart defects (CHDs) arise through various combinations of genetic and environmental factors. Our study explores how polymorphisms in the glutathione S-transferase (GST) genes affect the association between cigarette smoke exposure and CHDs. We analysed 299 mothers of children with CHDs and 284 mothers of children without any abnormalities who were recruited from six hospitals. The hair nicotine concentration (HNC) was used to quantify maternal smoke exposure, and the maternal GSTT1, and GSTM1 and GSTP1 genes were sequenced. We found a trend of higher adjusted odds ratios with higher maternal HNC levels, suggesting a dose-response relationship between maternal smoke exposure and CHDs. The lowest HNC range associated with an increased risk of CHDs was 0.213-0.319 ng/mg among the mothers with functional deletions of GSTM1 or GSTT1and 0.319-0.573 ng/mg among the mothers with normal copies of GSTM1 and GSTT1. In addition, the adjusted odds ratio for an HNC of >0.573 ng/mg was 38.53 among the mothers with the GSTP1 AG or GG genotype, which was 7.76 (χ(2) = 6.702, p = 0.010) times greater than the AOR in the mothers with GSTP1 AA genotype. Our study suggests that polymorphisms of maternal GST genes may modify the association of maternal smoke exposure with CHDs. PMID:26456689

  5. The Glutathione-S-Transferase, Cytochrome P450 and Carboxyl/Cholinesterase Gene Superfamilies in Predatory Mite Metaseiulus occidentalis

    PubMed Central

    Hoy, Marjorie A.

    2016-01-01

    Pesticide-resistant populations of the predatory mite Metaseiulus (= Typhlodromus or Galendromus) occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) have been used in the biological control of pest mites such as phytophagous Tetranychus urticae. However, the pesticide resistance mechanisms in M. occidentalis remain largely unknown. In other arthropods, members of the glutathione-S-transferase (GST), cytochrome P450 (CYP) and carboxyl/cholinesterase (CCE) gene superfamilies are involved in the diverse biological pathways such as the metabolism of xenobiotics (e.g. pesticides) in addition to hormonal and chemosensory processes. In the current study, we report the identification and initial characterization of 123 genes in the GST, CYP and CCE superfamilies in the recently sequenced M. occidentalis genome. The gene count represents a reduction of 35% compared to T. urticae. The distribution of genes in the GST and CCE superfamilies in M. occidentalis differs significantly from those of insects and resembles that of T. urticae. Specifically, we report the presence of the Mu class GSTs, and the J’ and J” clade CCEs that, within the Arthropoda, appear unique to Acari. Interestingly, the majority of CCEs in the J’ and J” clades contain a catalytic triad, suggesting that they are catalytically active. They likely represent two Acari-specific CCE clades that may participate in detoxification of xenobiotics. The current study of genes in these superfamilies provides preliminary insights into the potential molecular components that may be involved in pesticide metabolism as well as hormonal/chemosensory processes in the agriculturally important M. occidentalis. PMID:27467523

  6. Substrate Specificity Combined with Stereopromiscuity in Glutathione Transferase A4-4-dependent Metabolism of 4-Hydroxynonenal

    PubMed Central

    Balogh, Larissa M.; Le Trong, Isolde; Kripps, Kimberly A.; Shireman, Laura M.; Stenkamp, Ronald E.; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2010-01-01

    Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity towards them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is pre-organized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and an engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, co-crystallized with a GSH-HNE conjugate analog, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity towards a toxic racemate, concomitant with high catalytic efficiency and substrate specificity towards an endogenously formed toxin. PMID:20085333

  7. Substrate specificity combined with stereopromiscuity in glutathione transferase A4-4-dependent metabolism of 4-hydroxynonenal.

    PubMed

    Balogh, Larissa M; Le Trong, Isolde; Kripps, Kimberly A; Shireman, Laura M; Stenkamp, Ronald E; Zhang, Wei; Mannervik, Bengt; Atkins, William M

    2010-02-23

    Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity toward them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is preorganized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and an engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, cocrystallized with a GSH-HNE conjugate analogue, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead, the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity toward a toxic racemate, concomitant with high catalytic efficiency and substrate specificity toward an endogenously formed toxin. PMID:20085333

  8. Effect of glutathione-S-transferase polymorphisms on the cancer preventive potential of isothiocyanates: an epidemiological perspective.

    PubMed

    Seow, Adeline; Vainio, Harri; Yu, Mimi C

    2005-12-30

    Isothiocyanates (ITCs) are widely distributed in cruciferous vegetables and are biologically active against chemical carcinogenesis due to their ability to induce phase II conjugating enzymes. Among these is the glutathione-S-transferase (GST) family of enzymes, which in turn catalyzes the metabolism of ITCs, for which it has high substrate specificity. A recent body of epidemiologic data on the inverse association between cruciferous vegetable/ITC intake and cancers of the colo-rectum, lung and breast, also support that this protective effect is greater among individuals who possess the GSTM1 or T1 null genotype, and who would be expected to accumulate higher levels of ITC at the target tissue level, a pre-requisite for their enzyme-inducing effects. The association between ITC and cancer, and its modification by GST status, is most consistent for lung cancer and appears to be strongest among current smokers. Within limits, a comparison between groups which have been stratified by GST genotype may be less susceptible to confounding by other variables, given the random assortment of genes in gametogenesis. While a more complete understanding of the overall effects on health will need to take into account other components such as indoles and anti-oxidants, the interaction between ITC intake and GST genotype may provide a firmer basis to support a biologically significant role for ITC in cruciferous vegetables. PMID:16019037

  9. Pigs fed camelina meal increase hepatic gene expression of cytochrome 8b1, aldehyde dehydrogenase, and thiosulfate transferase

    PubMed Central

    2014-01-01

    Camelina sativa is an oil seed crop which can be grown on marginal lands. Camelina seed oil is rich in omega-3 fatty acids (>35%) and γ-tocopherol but is also high in erucic acid and glucosinolates. Camelina meal, is the by-product after the oil has been extracted. Camelina meal was fed to 28 d old weaned pigs at 3.7% and 7.4% until age 56 d. The camelina meal supplements in the soy based diets, improved feed efficiency but also significantly increased the liver weights. Gene expression analyses of the livers, using intra-species microarrays, identified increased expression of phase 1 and phase 2 drug metabolism enzymes. The porcine versions of the enzymes were confirmed by real time PCR. Cytochrome 8b1 (CYP8B1), aldehyde dehydrogenase 2 (Aldh2), and thiosulfate transferase (TST) were all significantly stimulated. Collectively, these genes implicate the camelina glucosinolate metabolite, methyl-sulfinyldecyl isothiocyanate, as the main xeniobiotic, causing increased hepatic metabolism and increased liver weight. PMID:24383433

  10. Proteomic Profiling of Cytosolic Glutathione Transferases from Three Bivalve Species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea

    PubMed Central

    Martins, José Carlos; Campos, Alexandre; Osório, Hugo; da Fonseca, Rute; Vasconcelos, Vítor

    2014-01-01

    Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs), in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST) were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties) between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism. PMID:24473139

  11. Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray.

    PubMed

    Shi, Jie; Sharif, Suhela; Ruijtenbeek, Rob; Pieters, Roland J

    2016-01-01

    O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity. PMID:26960196

  12. Mechanism of protective immunity by vaccination with recombinant Echinococcus granulosus glutathione S-transferase (Chinese strain) in mice

    PubMed Central

    ZHU, MINGXING; WANG, XIUQING; WANG, HAO; WANG, ZHISHENG; ZHAO, JIAQING; WANG, YANA; ZHAO, WEI

    2015-01-01

    The aim of this study was to investigate the immunoprotective effects of recombinant Echinococcus granulosus glutathione S-transferase (rEgGST) against the development of protoscolices (PSCs), and to determine the mechanisms underlying this protection. ICR mice were subcutaneously immunized three times with rEgGST at weeks 0, 2 and 4, followed by the intraperitoneal administration of E. granulosus PSCs at week 10. Six mice in each group were sacrificed at 0, 2, 4, 6, 10, 18 and 30 weeks following the initial vaccination in order to observe the macroscopic and microscopic effects of parasite development. Various analyses were subsequently conducted, including determination of the levels of immunoglobulins (Igs) and cytokines. Significant differences were observed a number of indices of immune response following immunization with rEgGST. These included reduced cyst formation and elevated levels of IgG1, IgG2a, IgG3, IL-2, IL-4, IL-10 and IFN-γ, which indicated an increased percentage of immune helper cells. The results of the present study suggest that immunization with rEgGST in mice is able to successfully reduce the PSC-induced formation of cysts and to stimulate an immune response, suggesting that rEgGST possesses potential value as a candidate vaccine for PSC infection. PMID:26622451

  13. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells

    PubMed Central

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-01-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3′-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA–protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1–TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  14. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells.

    PubMed

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-02-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3'-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA-protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1-TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  15. H.p.l.c. separation and study of the charge isomers of human placental glutathione transferase.

    PubMed Central

    Radulovic, L L; Kulkarni, A P

    1986-01-01

    Glutathione transferase (GST) from human placenta was purified by affinity chromatography and anion-exchange h.p.l.c. The enzyme exhibited different chromatographic and electrophoretic behaviours according to the concentration of GSH, suggesting a possible change in the net charge of the molecule and a concomitant conformational change due to ligand binding. Two interconvertible forms were quantitatively separated into distinct catalytically active states by h.p.l.c. Depending upon the GSH concentration, polyacrylamide-gel electrophoresis revealed the presence of one or two bands. A Kd of 0.42 mM for GSH was determined fluorimetrically. The loss in intrinsic fluorescence also suggested a conformational change in the enzyme. Kinetic studies using ethacrynic acid were conducted to determine whether the presumed conformational change could effect the catalytic capability of placental GST. A biphasic response in initial velocities was observed with increasing concentrations of GSH. Two apparent Km values of 0.38 and 50.27 mM were obtained for GSH, whereas Vmax. values showed a 46-fold difference. It was concluded that the enzyme assumes a highly anionic form in the presence of a low GSH concentration, whereas it is converted into relatively weaker anionic form when its immediate environment contains a high GSH concentration. Since the average tissue concentration of total GSH was estimated at 0.11 mM for term placenta, the results suggest that the high-affinity-low-activity conformer would predominate in vivo. Images Fig. 2. PMID:3800986

  16. m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts.

    PubMed

    Liu, Y; Santi, D V

    2000-07-18

    A family of RNA m(5)C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m(5)C 967 MTase, Fmu, as an initial probe. The RNA m(5)C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m(5)C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m(5)U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m(5)C MTase uses a different Cys as a catalytic nucleophile than the DNA m(5)C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m(5)C MTases remains unknown. PMID:10899996

  17. Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins

    PubMed Central

    Trapannone, Riccardo; Mariappa, Daniel; Ferenbach, Andrew T.; vanAalten, Daan M.F.

    2016-01-01

    O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome. PMID:27048592

  18. The two paralogue phoN (phosphinothricin acetyl transferase) genes of Pseudomonas putida encode functionally different proteins.

    PubMed

    Páez-Espino, A David; Chavarría, Max; de Lorenzo, Víctor

    2015-09-01

    Phosphinothricin (PPT) is a non-specific inhibitor of glutamine synthetase that has been employed as herbicide for selection of transgenic plants expressing cognate resistance genes. While the soil bacterium Pseudomonas putida KT2440 has been generally considered PPT-sensitive, inspection of its genome sequence reveals the presence of two highly similar open reading frames (PP_1924 and PP_4846) encoding acetylases with a potential to cause tolerance to the herbicide. To explore this possibility, each of these genes (named phoN1 and phoN2) was separately cloned and their activities examined in vivo and in vitro. Genetic and biochemical evidence indicated that phoN1 encodes a bona fide PPT-acetyl transferase, the expression of which suffices to make P. putida tolerant to high concentrations of the herbicide. In contrast, PhoN2 does not act on PPT but displays instead activity against methionine sulfoximine (MetSox), another glutamine synthetase inhibitor. When the geometry of the substrate-binding site of PhoN1 was grafted with the equivalent residues of the predicted PhoN2 structure, the resulting protein increased significantly MetSox resistance of the expression host concomitantly with the loss of activity on PPT. These observations uncover intricate biochemical and genetic interactions among soil microorganisms and how they can be perturbed by exposure to generic herbicides in soil. PMID:25684119

  19. Nuclear glutathione S-transferase pi prevents apoptosis by reducing the oxidative stress-induced formation of exocyclic DNA products.

    PubMed

    Kamada, Kensaku; Goto, Shinji; Okunaga, Tomohiro; Ihara, Yoshito; Tsuji, Kentaro; Kawai, Yoshichika; Uchida, Koji; Osawa, Toshihiko; Matsuo, Takayuki; Nagata, Izumi; Kondo, Takahito

    2004-12-01

    We previously found that nuclear glutathione S-transferase pi (GSTpi) accumulates in cancer cells resistant to anticancer drugs, suggesting that it has a role in the acquisition of resistance to anticancer drugs. In the present study, the effect of oxidative stress on the nuclear translocation of GSTpi and its role in the protection of DNA from damage were investigated. In human colonic cancer HCT8 cells, the hydrogen peroxide (H(2)O(2))-induced increase in nuclear condensation, the population of sub-G(1) peak, and the number of TUNEL-positive cells were observed in cells pretreated with edible mushroom lectin, an inhibitor of the nuclear transport of GSTpi. The DNA damage and the formation of lipid peroxide were dependent on the dose of H(2)O(2) and the incubation time. Immunological analysis showed that H(2)O(2) induced the nuclear accumulation of GSTpi but not of glutathione peroxidase. Formation of the 7-(2-oxo-hepyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct by the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2'-deoxyguanosine was inhibited by GSTpi in the presence of glutathione. The conjugation product of 4-oxo-2-nonenal, a lipid aldehyde of 13-HPODE, with GSH in the presence of GSTpi, was identified by LS/MS. These results suggested that nuclear GSTpi prevents H(2)O(2)-induced DNA damage by scavenging the formation of lipid-peroxide-modified DNA. PMID:15528046

  20. The Plasmodium palmitoyl-S-acyl-transferase DHHC2 is essential for ookinete morphogenesis and malaria transmission.

    PubMed

    Santos, Jorge M; Kehrer, Jessica; Franke-Fayard, Blandine; Frischknecht, Friedrich; Janse, Chris J; Mair, Gunnar R

    2015-01-01

    The post-translational addition of C-16 long chain fatty acids to protein cysteine residues is catalysed by palmitoyl-S-acyl-transferases (PAT) and affects the affinity of a modified protein for membranes and therefore its subcellular localisation. In apicomplexan parasites this reversible protein modification regulates numerous biological processes and specifically affects cell motility, and invasion of host cells by Plasmodium falciparum merozoites and Toxoplasma gondii tachyzoites. Using inhibitor studies we show here that palmitoylation is key to transformation of zygotes into ookinetes during initial mosquito infection with P. berghei. We identify DHHC2 as a unique PAT mediating ookinete formation and morphogenesis. Essential for life cycle progression in asexual blood stage parasites and thus refractory to gene deletion analyses, we used promoter swap (ps) methodology to maintain dhhc2 expression in asexual blood stages but down regulate expression in sexual stage parasites and during post-fertilization development of the zygote. The ps mutant showed normal gamete formation, fertilisation and DNA replication to tetraploid cells, but was characterised by a complete block in post-fertilisation development and ookinete formation. Our report highlights the crucial nature of the DHHC2 palmitoyl-S-acyltransferase for transmission of the malaria parasite to the mosquito vector through its essential role for ookinete morphogenesis. PMID:26526684

  1. Fractal binding and dissociation kinetics of lecithin cholesterol acyl transferase (LCAT), a heart-related compound, on biosensor surfaces

    NASA Astrophysics Data System (ADS)

    Doke, Atul M.; Sadana, Ajit

    2006-05-01

    A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg-white apoA-I rHDL immobilized on a biosensor chip surface.1 Single- and dual- fractal models were employed to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (Corel Corporation Limited).2 The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution, and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.

  2. A novel glutathione-S transferase immunosensor based on horseradish peroxidase and double-layer gold nanoparticles.

    PubMed

    Lu, Dingqiang; Lu, Fuping; Pang, Guangchang

    2016-06-01

    GSTs, a biotransformation enzyme group, can perform metabolism, drug transfer and detoxification functions. Rapid detection of the GSTs with more sensitive approaches is of great importance. In the current study, a novel double-layer gold nanoparticles-electrochemical immunosensor electrode (DGN-EIE) immobilized with Glutathione S-Transferase (GST) antibody derived from Balb/c mice was developed. To increase the fixed quantity of antibodies and electrochemical signal, an electrochemical biosensing signal amplification system was utilized with gold nanoparticles-thionine-chitosan absorbing horseradish peroxidase (HRP). In addition, transmission electron microscope (TEM) was used to characterize the nanogold solution. To evaluate the quality of DGN-EIE, the amperometric I-t curve method was applied to determine the GST in PBS. The results showed that the response current had a good linear correlation with the GST concentration ranged from 0.1-10(4) pg/mL. The lowest detection limit was found at 0.03 pg/mL(S/N = 3). The linear equation was deduced as △I/% = 7.386lgC + 22.36 (R(2) = 0.998). Moreover, it was validated with high sensitivity and reproducibility. Apparently, DGN-EIE may be a very useful tool for monitoring the GST. PMID:27220630

  3. The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferases in zebrafish (Danio rerio) livers.

    PubMed

    Dong, Miao; Zhu, Lusheng; Shao, Bo; Zhu, Shaoyuan; Wang, Jun; Xie, Hui; Wang, Jinhua; Wang, Fenghua

    2013-06-01

    Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10μgL(-1)) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver. PMID:23523001

  4. Overexpression of Glutathione Transferase E7 in Drosophila Differentially Impacts Toxicity of Organic Isothiocyanates in Males and Females

    PubMed Central

    Mannervik, Bengt; Mannervik, Mattias

    2014-01-01

    Organic isothiocyanates (ITCs) are allelochemicals produced by plants in order to combat insects and other herbivores. The compounds are toxic electrophiles that can be inactivated and conjugated with intracellular glutathione in reactions catalyzed by glutathione transferases (GSTs). The Drosophila melanogaster GSTE7 was heterologously expressed in Escherichia coli and purified for functional studies. The enzyme showed high catalytic activity with various isothiocyanates including phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), which in millimolar dietary concentrations conferred toxicity to adult D. melanogaster leading to death or a shortened life-span of the flies. In situ hybridization revealed a maternal contribution of GSTE7 transcripts to embryos, and strongest zygotic expression in the digestive tract. Transgenesis involving the GSTE7 gene controlled by an actin promoter produced viable flies expressing the GSTE7 transcript ubiquitously. Transgenic females show a significantly increased survival when subjected to the same PEITC treatment as the wild-type flies. By contrast, transgenic male flies show a significantly lower survival rate. Oviposition activity was enhanced in transgenic flies. The effect was significant in transgenic females reared in the absence of ITCs as well as in the presence of 0.15 mM PEITC or 1 mM AITC. Thus the GSTE7 transgene elicits responses to exposure to ITC allelochemicals which differentially affect life-span and fecundity of male and female flies. PMID:25329882

  5. Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins.

    PubMed

    Trapannone, Riccardo; Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

    2016-06-15

    O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome. PMID:27048592

  6. Immunocytochemical studies of the distribution of alpha and pi isoforms of glutathione S-transferase in cystic renal diseases.

    PubMed

    Hiley, C G; Otter, M; Bell, J; Strange, R C; Keeling, J W

    1994-01-01

    We describe immunohistochemical studies of the expression of alpha and pi class glutathione S-transferases (GSTs) in normal fetal kidneys. These define, in greater detail, changes in expression of alpha isoforms in the proximal tubule. At about 36 weeks of gestation expression of alpha isoforms was down-regulated in the distal tubules and collecting ducts while pi was expressed throughout the nephron. Tubular expression of alpha isoforms was restricted to the part adjacent to the glomerulus; cells farthest from the glomerulus were negative. After 40 weeks of gestation, alpha isoforms were expressed along the entire proximal tubule, while pi was restricted to the distal tubule and collecting ducts. GST expression was also studied in multicystic renal dysplasia, autosomal recessive polycystic kidney disease, and autosomal dominant polycystic kidney disease to determine whether the patterns of expression of alpha and pi isoforms allow identification of the origin of the cysts that characterize these diseases. Cysts were lined by epithelia that were strongly positive for alpha and pi isoforms. The epithelia of noncystic nephrons in renal cystic dysplasia demonstrated delayed maturity, suggesting that GST expression was dependent on the stage of development and not length of gestation. PMID:8066005

  7. The Putative O-Linked N-Acetylglucosamine Transferase SPINDLY Inhibits Class I TCP Proteolysis to Promote Sensitivity to Cytokinin.

    PubMed

    Steiner, Evyatar; Livne, Sivan; Kobinson-Katz, Tammy; Tal, Lior; Pri-Tal, Oded; Mosquna, Assaf; Tarkowská, Danuše; Mueller, Bruno; Tarkowski, Petr; Weiss, David

    2016-06-01

    Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers. PMID:27208284

  8. Identification of Putative Carboxylesterase and Glutathione S-transferase Genes from the Antennae of the Chilo suppressalis (Lepidoptera: Pyralidae)

    PubMed Central

    Liu, Su; Gong, Zhong-Jun; Rao, Xiang-Jun; Li, Mao-Ye; Li, Shi-Guang

    2015-01-01

    In insects, rapid degradation of odorants in antennae is extremely important for the sensitivity of olfactory receptor neurons. Odorant degradation in insect antennae is mediated by multiple enzymes, especially the carboxylesterases (CXEs) and glutathione S-transferases (GSTs). The Asiatic rice borer, Chilo suppressalis, is an economically important lepidopteran pest which causes great economic damage to cultivated rice crops in many Asian countries. In this study, we identified 19 putative CXE and 16 GST genes by analyzing previously constructed antennal transcriptomes of C. suppressalis. BLASTX best hit results showed that these genes are most homologous to their respective orthologs in other lepidopteran species. Phylogenetic analyses revealed that these CXE and GST genes were clustered into various clades. Reverse-transcription quantitative polymerase chain reaction assays showed that three CXE genes (CsupCXE8, CsupCXE13, and CsupCXE18) are antennae-enriched. These genes are candidates for involvement in odorant degradation. Unexpectedly, none of the GST genes were found to be antennae-specific. Our results pave the way for future researches of the odorant degradation mechanism of C. suppressalis at the molecular level. PMID:26198868

  9. Induction of carnitine palmitoyl transferase 1 and fatty acid oxidation by retinoic acid in HepG2 cells.

    PubMed

    Amengual, Jaume; Petrov, Petar; Bonet, M Luisa; Ribot, Joan; Palou, Andreu

    2012-11-01

    The vitamin A derivative retinoic acid (RA) is an important regulator of mammalian adiposity and lipid metabolism, primarily acting at the gene expression level through nuclear receptors of the RA receptor (RAR) and retinoid X receptor (RXR) subfamilies. Here, we studied cell-autonomous effects of RA on fatty acid metabolism, particularly fatty acid oxidation, in human hepatoma HepG2 cells. Exposure to all-trans RA (ATRA) up-regulated the expression of carnitine palmitoyl transferase-1 (CPT1-L) in HepG2 cells in a dose- and time-dependent manner, and increased cellular oxidation rate of exogenously added radiolabeled palmitate. The effect of ATRA on gene expression of CPT1-L was: dependent on ongoing transcription, reproduced by both 9-cis RA and a pan-RXR agonist (but not a pan-RAR agonist) and abolished following RXRα partial siRNA-mediated silencing. CPT1-L gene expression was synergistically induced in HepG2 cells simultaneously exposed to ATRA and a selective peroxisome proliferator-activated receptor α agonist. We conclude that ATRA treatment enhances fatty acid catabolism in hepatocytes through RXR-mediated mechanisms that likely involve the transactivation of the PPARα:RXR heterodimer. Knowledge of agents and nutrient-derivatives capable of enhancing substrate oxidation systemically and specifically in liver, and their mechanisms of action, may contribute to new avenues of prevention and treatment of fatty liver, obesity and other metabolic syndrome-related disorders. PMID:22871568

  10. Tumor efficacy and bone marrow-sparing properties of TER286, a cytotoxin activated by glutathione S-transferase.

    PubMed

    Morgan, A S; Sanderson, P E; Borch, R F; Tew, K D; Niitsu, Y; Takayama, T; Von Hoff, D D; Izbicka, E; Mangold, G; Paul, C; Broberg, U; Mannervik, B; Henner, W D; Kauvar, L M

    1998-06-15

    TER286 is a latent drug activated by human glutathione S-transferase (GST) isoforms P1-1 and A1-1 to produce a nitrogen mustard alkylating agent. M7609 human colon carcinoma, selected for resistance to doxorubicin, and MCF-7 human breast carcinoma, selected for resistance to cyclophosphamide, both showed increased sensitivity to TER286 over their parental lines in parallel with increased expression of GST P1-1. In primary human tumor clonogenic assays, the spectrum of cytotoxic activity observed for TER286 was both broad and unusual when compared to a variety of current drugs. In murine xenografts of M7609 engineered to have high, medium, or low GST P1-1, responses to TER286 were positively correlated with the level of P1-1. Cytotoxicity was also observed in several other cell culture and xenograft models. In xenografts of the MX-1 human breast carcinoma, tumor growth inhibition or regression was observed in nearly all of the animals treated with an aggressive regimen of five daily doses. This schedule resulted in a 24-h posttreatment decline in bone marrow progenitors to 60% of control and was no worse than for a single dose of TER286. These studies have motivated election of TER286 as a clinical candidate. PMID:9635580

  11. m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

    PubMed Central

    Liu, Yaoquan; Santi, Daniel V.

    2000-01-01

    A family of RNA m5C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m5C 967 MTase, Fmu, as an initial probe. The RNA m5C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m5C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m5U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m5C MTase uses a different Cys as a catalytic nucleophile than the DNA m5C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m5C MTases remains unknown. PMID:10899996

  12. Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole in vitro.

    PubMed

    Hansen, Tina V A; Fryganas, Christos; Acevedo, Nathalie; Caraballo, Luis; Thamsborg, Stig M; Mueller-Harvey, Irene; Williams, Andrew R

    2016-08-01

    Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity. PMID:27094225

  13. Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray

    PubMed Central

    Shi, Jie; Sharif, Suhela; Ruijtenbeek, Rob; Pieters, Roland J.

    2016-01-01

    O-GlcNAcylation is a reversible and dynamic protein post-translational modification in mammalian cells. The O-GlcNAc cycle is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays important role in many vital cellular events including transcription, cell cycle regulation, stress response and protein degradation, and altered O-GlcNAcylation has long been implicated in cancer, diabetes and neurodegenerative diseases. Recently, numerous approaches have been developed to identify OGT substrates and study their function, but there is still a strong demand for highly efficient techniques. Here we demonstrated the utility of the peptide microarray approach to discover novel OGT substrates and study its specificity. Interestingly, the protein RBL-2, which is a key regulator of entry into cell division and may function as a tumor suppressor, was identified as a substrate for three isoforms of OGT. Using peptide Ala scanning, we found Ser 420 is one possible O-GlcNAc site in RBL-2. Moreover, substitution of Ser 420, on its own, inhibited OGT activity, raising the possibility of mechanism-based development for selective OGT inhibitors. This approach will prove useful for both discovery of novel OGT substrates and studying OGT specificity. PMID:26960196

  14. Conserved Nutrient Sensor O-GlcNAc Transferase Is Integral to C. elegans Pathogen-Specific Immunity

    PubMed Central

    Bond, Michelle R.; Ghosh, Salil K.; Wang, Peng; Hanover, John A.

    2014-01-01

    Discriminating pathogenic bacteria from bacteria used as a food source is key to Caenorhabidits elegans immunity. Using mutants defective in the enzymes of O-linked N-acetylglucosamine (O-GlcNAc) cycling, we examined the role of this nutrient-sensing pathway in the C. elegans innate immune response. Genetic analysis showed that deletion of O-GlcNAc transferase (ogt-1) yielded animals hypersensitive to the human pathogen S. aureus but not to P. aeruginosa. Genetic interaction studies revealed that nutrient-responsive OGT-1 acts through the conserved β-catenin (BAR-1) pathway and in concert with p38 MAPK (PMK-1) to modulate the immune response to S. aureus. Moreover, whole genome transcriptional profiling revealed that O-GlcNAc cycling mutants exhibited deregulation of unique stress- and immune-responsive genes. The participation of nutrient sensor OGT-1 in an immunity module evolutionarily conserved from C. elegans to humans reveals an unexplored nexus between nutrient availability and a pathogen-specific immune response. PMID:25474640

  15. Glutathione S-transferase SlGSTE1 in Spodoptera litura may be associated with feeding adaptation of host plants.

    PubMed

    Zou, Xiaopeng; Xu, Zhibin; Zou, Haiwang; Liu, Jisheng; Chen, Shuna; Feng, Qili; Zheng, Sichun

    2016-03-01

    Spodoptera litura is polyphagous pest insect and feeds on plants of more than 90 families. In this study the role of glutathione S-transferase epilson 1 (slgste1) in S. litura in detoxification was examined. This gene was up-regulated in the midgut of S. litura at the transcriptional and protein levels when the insect fed on Brassica juncea or diet containing phytochemicals such as indole-3-carbinol and allyl-isothiocyanate that are metabolic products of sinigrin and glucobrassicin in B. juncea. The SlGSTE1 could catalyze the conjugation of reduced glutathione and indole-3-carbinol and allyl-isothiocyanate, as well as xanthotoxin, which is a furanocoumarin, under in vitro condition. When the expression of Slgste1 in the larvae was suppressed with RNAi, the larval growth and feeding rate were decreased. Furthermore, the up-regulated expression of the SlGSTE1 protein in the midgut of larvae that fed on different host plants was detected by 2-DE and ESI/MS analysis. The feeding adaptation from the most to the least of the larvae for the various host plants was Brassica alboglabra, Brassica linn. Pekinensis, Cucumis sativus, Ipomoea batatas, Arachis hypogaea and Capsicum frutescens. All the results together suggest that Slgste1 is a critical detoxifying enzyme that is induced by phytochmicals in the host plants and, inter alia, may be related to host plant adaptation of S. litura. PMID:26631599

  16. Glutathione S-Transferase of Brown Planthoppers (Nilaparvata lugens) Is Essential for Their Adaptation to Gramine-Containing Host Plants

    PubMed Central

    Yu, Jing-Ya; Jin, Yu; Ling, Bing; Du, Jin-Ping; Li, Gui-Hua; Qin, Qing-Ming; Cai, Qing-Nian

    2013-01-01

    Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa), a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs) is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms. PMID:23700450

  17. Genome-Wide Analysis of the Glutathione S-Transferase Gene Family in Capsella rubella: Identification, Expression, and Biochemical Functions

    PubMed Central

    He, Gang; Guan, Chao-Nan; Chen, Qiang-Xin; Gou, Xiao-Jun; Liu, Wei; Zeng, Qing-Yin; Lan, Ting

    2016-01-01

    Extensive subfunctionalization might explain why so many genes have been maintained after gene duplication, which provides the engine for gene family expansion. However, it is still a particular challenge to trace the evolutionary dynamics and features of functional divergences in a supergene family over the course of evolution. In this study, we identified 49 Glutathione S-transferase (GST) genes from the Capsella rubella, a close relative of Arabidopsis thaliana and a member of the mustard family. Capsella GSTs can be categorized into eight classes, with tau and phi GSTs being the most numerous. The expansion of the two classes mainly occurs through tandem gene duplication, which results in tandem-arrayed gene clusters on chromosomes. By integrating phylogenetic analysis, expression patterns, and biochemical functions of Capsella and Arabidopsis GSTs, functional divergence, both in gene expression and enzymatic properties, were clearly observed in paralogous gene pairs in Capsella (even the most recent duplicates), and orthologous GSTs in Arabidopsis/Capsella. This study provides functional evidence for the expansion and organization of a large gene family in closely related species.

  18. Pigs fed camelina meal increase hepatic gene expression of cytochrome 8b1, aldehyde dehydrogenase, and thiosulfate transferase.

    PubMed

    Meadus, William Jon; Duff, Pascale; McDonald, Tanya; Caine, William R

    2014-01-01

    Camelina sativa is an oil seed crop which can be grown on marginal lands. Camelina seed oil is rich in omega-3 fatty acids (>35%) and γ-tocopherol but is also high in erucic acid and glucosinolates. Camelina meal, is the by-product after the oil has been extracted. Camelina meal was fed to 28 d old weaned pigs at 3.7% and 7.4% until age 56 d. The camelina meal supplements in the soy based diets, improved feed efficiency but also significantly increased the liver weights. Gene expression analyses of the livers, using intra-species microarrays, identified increased expression of phase 1 and phase 2 drug metabolism enzymes. The porcine versions of the enzymes were confirmed by real time PCR. Cytochrome 8b1 (CYP8B1), aldehyde dehydrogenase 2 (Aldh2), and thiosulfate transferase (TST) were all significantly stimulated. Collectively, these genes implicate the camelina glucosinolate metabolite, methyl-sulfinyldecyl isothiocyanate, as the main xeniobiotic, causing increased hepatic metabolism and increased liver weight. PMID:24383433

  19. A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology, Stress Tolerance, and Pathogenic Outcome

    PubMed Central

    Santiago-Tirado, Felipe H.; Peng, Tao; Yang, Meng; Hang, Howard C.; Doering, Tamara L.

    2015-01-01

    Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter’s opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT), one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics. PMID:25970403

  20. Proteomic profiling of cytosolic glutathione transferases from three bivalve species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea.

    PubMed

    Martins, José Carlos; Campos, Alexandre; Osório, Hugo; da Fonseca, Rute; Vasconcelos, Vítor

    2014-01-01

    Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs), in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST) were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties) between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism. PMID:24473139

  1. A glutathione S-transferase inducer from papaya: rapid screening, identification and structure-activity relationship of isothiocyanates.

    PubMed

    Nakamura, Y; Morimitsu, Y; Uzu, T; Ohigashi, H; Murakami, A; Naito, Y; Nakagawa, Y; Osawa, T; Uchida, K

    2000-09-01

    We have developed a simple system for rapid detection and measurement of glutathione S-transferase placental form (GSTP1) that detoxify polycyclic aromatic hydrocarbons using the cultured rat normal liver epithelial cell line, (RL34) cells. Survey of fruit extracts for GST inducing ability identified both papaya and avocado as significant sources. Benzyl isothiocyanate (BITC) was isolated from papaya methanol extract as a principal inducer of GST activity. Further, the GST inducing ability of a total of 20 isothiocyanates (ITCs) and their derivatives was investigated. Some ITCs showed significant induction, and BITC was one of the most potent inducers among all compounds tested in the present study. The modification of isothiocyanate group (-NCS) or introduction of substituent group to the alpha-carbon modifies GST induction. Moreover, a significant correlation (P<0.01, r=0.913) between the GST activity enrichment and GSTP1 protein induction by ITCs was observed. We also indicated that phenethyl ITC and nitrophenyl ITC, potently inducing GST activity, but not inactive benzyl isocyanate, are potential inducers of intracellular reactive oxygen intermediates (ROIs). Our system of GSTP1 induction is appropriate for the chemical research such as screening and identification of novel type of inducers as well as the structure-activity relationship studies, providing mechanistic insight into essential structural elements for GSTP1 induction. PMID:10936680

  2. Protein isoprenylation regulates osteogenic differentiation of mesenchymal stem cells: effect of alendronate, and farnesyl and geranylgeranyl transferase inhibitors

    PubMed Central

    Duque, G; Vidal, C; Rivas, D

    2011-01-01

    BACKGROUND AND PURPOSE Protein isoprenylation is an important step in the intracellular signalling pathway conducting cell growth and differentiation. In bone, protein isoprenylation is required for osteoclast differentiation and activation. However, its role in osteoblast differentiation and function remains unknown. In this study, we assessed the role of protein isoprenylation in osteoblastogenesis in a model of mesenchymal stem cells (MSC) differentiation. EXPERIMENTAL APPROACH We tested the effect of an inhibitor of farnesylation [farnesyl transferase inhibitor-277 (FTI-277)] and one of geranylgeranylation [geranylgeranyltransferase inhibitor-298 (GGTI-298)] on osteoblast differentiating MSC. In addition, we tested the effect of alendronate on protein isoprenylation in this model either alone or in combination with other inhibitors of isoprenylation. KEY RESULTS Initially, we found that levels of unfarnesylated proteins (prelamin A and HDJ-2) increased after treatment with FTI-277 concomitantly affecting osteoblastogenesis and increasing nuclear morphological changes without affecting cell survival. Furthermore, inhibition of geranylgeranylation by GGTI-298 alone increased osteoblastogenesis. This effect was enhanced by the combination of GGTI-298 and alendronate in the osteogenic media. CONCLUSIONS AND IMPLICATIONS Our data indicate that both farnesylation and geranylgeranylation play a role in osteoblastogenesis. In addition, a new mechanism of action for alendronate on protein isoprenylation in osteogenic differentiating MSC in vitro was found. In conclusion, protein isoprenylation is an important component of the osteoblast differentiation process that could constitute a new therapeutic target for osteoporosis in the future. PMID:21077849

  3. Abamectin resistance in strains of vegetable leafminer, Liriomyza sativae (Diptera: Agromyzidae) is linked to elevated glutathione S-transferase activity.

    PubMed

    Wei, Qing-Bo; Lei, Zhong-Ren; Nauen, Ralf; Cai, Du-Cheng; Gao, Yu-Lin

    2015-04-01

    Abamectin resistance was selected in the vegetable leafminer, Liriomyza sativae (Blanchard) (Diptera: Agromyzidae) under laboratory conditions, and cross-resistance patterns and possible resistance mechanisms in the abamectin-resistant strains (AL-R, AF-R) were investigated. Compared with the susceptible strain (SS), strain AL-R displayed 39-fold resistance to abamectin after 20 selection cycles during 25 generations, and strain AF-R exhibited 59-fold resistance to abamectin after 16 selection cycles during 22 generations. No cross-resistance to cyromazine was found in both abamectin-resistant strains. However, we failed to select for cyromazine resistance in L. sativae under laboratory conditions by conducting 17 selection cycles during 22 generations. However, moderate levels of cross-resistance to abamectin (6-9 fold) were observed in strains which received cyromazine treatments. Biochemical analysis showed that glutathione S-transferase (GST) activity in both abamectin-resistant strains (AL-R, AF-R) was significantly higher than in the susceptible strain (SS), suggesting metabolically driven resistance to abamectinin L. sativae. Recommendations of mixtures or rotation of cyromazine and abamectin should be considered carefully, as consecutive cyromazine treatments may select for low-level cross-resistance to abamectin. PMID:25813391

  4. The catechol-O-methyl transferase Val158Met polymorphism and experience of reward in the flow of daily life.

    PubMed

    Wichers, Marieke; Aguilera, Mari; Kenis, Gunter; Krabbendam, Lydia; Myin-Germeys, Inez; Jacobs, Nele; Peeters, Frenk; Derom, Catherine; Vlietinck, Robert; Mengelers, Ron; Delespaul, Philippe; van Os, Jim

    2008-12-01

    Genetic moderation of experience of reward in response to environmental stimuli is relevant for the study of many psychiatric disorders. Experience of reward, however, is difficult to capture, as it involves small fluctuations in affect in response to small events in the flow of daily life. This study examined a momentary assessment reward phenotype in relation to the catechol-O-methyl transferase (COMT) Val(158)Met polymorphism. A total of 351 participants from a twin study participated in an Experience Sampling Method procedure to collect daily life experiences concerning events, event appraisals, and affect. Reward experience was operationalized, as the effect of event appraisal on positive affect (PA). Associations between COMT Val(158)Met genotype and event appraisal on the one hand and PA on the other were examined using multilevel random regression analysis. Ability to experience reward increased with the number of 'Met' alleles of the subject, and this differential effect of genotype was greater for events that were experienced as more pleasant. The effect size of genotypic moderation was quite large: subjects with the Val/Val genotype generated almost similar amounts of PA from a 'very pleasant event' as Met/Met subjects did from a 'bit pleasant event'. Genetic variation with functional impact on cortical dopamine tone has a strong influence on reward experience in the flow of daily life. Genetic moderation of ecological measures of reward experience is hypothesized to be of major relevance to the development of various behavioral disorders, including depression and addiction. PMID:17687265

  5. The ribonucleotidyl transferase USIP-1 acts with SART3 to promote U6 snRNA recycling

    PubMed Central

    Rüegger, Stefan; Miki, Takashi S.; Hess, Daniel; Großhans, Helge

    2015-01-01

    The spliceosome is a large molecular machine that serves to remove the intervening sequences that are present in most eukaryotic pre-mRNAs. At its core are five small nuclear ribonucleoprotein complexes, the U1, U2, U4, U5 and U6 snRNPs, which undergo dynamic rearrangements during splicing. Their reutilization for subsequent rounds of splicing requires reversion to their original configurations, but little is known about this process. Here, we show that ZK863.4/USIP-1 (U Six snRNA-Interacting Protein-1) is a ribonucleotidyl transferase that promotes accumulation of the Caenorhabditis elegans U6 snRNA. Endogenous USIP-1–U6 snRNA complexes lack the Lsm proteins that constitute the protein core of the U6 snRNP, but contain the U6 snRNP recycling factor SART3/B0035.12. Furthermore, co-immunoprecipitation experiments suggest that SART3 but not USIP-1 occurs also in a separate complex containing both the U4 and U6 snRNPs. Based on this evidence, genetic interaction between usip-1 and sart-3, and the apparent dissociation of Lsm proteins from the U6 snRNA during spliceosome activation, we propose that USIP-1 functions upstream of SART3 to promote U6 snRNA recycling. PMID:25753661

  6. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

    PubMed Central

    Lira-Navarrete, Erandi; de las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

    2015-01-01

    Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans. PMID:25939779

  7. Mouse model for somatic mutation at the HPRT (hypoxanthine phosphoribosyl-transferase) gene: Molecular and cellular analyses

    SciTech Connect

    Burkhart-Schultz, K.; Strout, C.L.; Jones, I.M.

    1989-07-11

    Our goal is to use the mouse to model the organismal, cellular and molecular factors that affect somatic mutagenesis in vivo. A fundamental tenet of genetic toxicology is that the principles of mutagenesis identified in one system can be used to predict the principles of mutagenesis in another system. The validity of this tenet depends upon the comparability of the systems involved. To begin to achieve an understanding of somatic mutagenesis in vivo, we have been studying mutations that occur in the hypoxanthine phosphoribosyl-transferase (HPRT) gene of lymphocytes of mice. Our in vivo model for somatic mutation allows us to analyse factors that affect somatic mutation. Having chosen the mouse, we are working with cells in which the karyotype is normal, and metabolic and DNA repair capacity are defined by the mouse strain chosen. At the organismal level, we can vary sex, age, the exposure history, and the tissue source of cells analysed. (All studies reported here have, however, used male mice.) At the cellular level, T lymphocytes and their precursors are the targets and reporters of mutation. 26 refs., 1 fig., 1 tab.

  8. Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5'-phosphate synthesis and pathogenesis of Magnaporthe oryzae.

    PubMed

    Qi, Zhongqiang; Liu, Muxing; Dong, Yanhan; Yang, Jie; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2016-04-01

    Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5'-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae. PMID:26810198

  9. Radioprotector WR1065 reduces radiation-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells

    SciTech Connect

    Grdina, D.J.; Hill, C.K.; Peraino, C. ); Biserka, N. ); Wells, R.L. . Dept. of Radiology and Radiation Biology)

    1985-06-01

    N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065) protects against radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster lung fibroblast cells. WR1065 (4 mm) was found to be effective in protecting against radiation-induced cell lethality only if present during irradiation. No protective effect was observed if the protector was added within 5 min after irradiation or 3 h later. The effect of WR1065 on radiation-induced mutation, expressed as resistance to the cytotoxic purine analogue 6-thioguanine (HGPRT), was also investigated. This agent was effective in reducing radiation-induced mutations regardless of when it was administered. Following 10 Gy of /sup 60/Co ..gamma..-rays, the mutation frequencies observed per 10/sup 6/ survivors were 77 +- 8, 27 +- 6, 42 +- 7, and 42 +- 7 for radiation only, and WR1065 present during, immediately after, or 3 h after irradiation. These data suggest that although a segment of radiation-induced damage leading to reproductive death cannot be modulated through the postirradiation action of WR1065, processes leading to the fixation of gross genetic damage and mutation induction in surviving cells can be effectively altered and interfered with leading to a marked reduction in mutation frequency.

  10. Potential use of acetylcholinesterase, glutathione-S-transferase and metallothionein for assessment of contaminated sediment in tropical chironomid, Chironomus javanus.

    PubMed

    Somparn, A; Iwai, C B; Noller, B

    2015-11-01

    Heavy metals and organophosphorus insecticide is known to act as disruptors for the enzyme system, leading to physiologic disorders. The present study was conducted to investigate the potential use of these enzymes as biomarkers in assessment of contaminated sediments on tropical chironomid species. Acetylcholinesterase (AChE), glutathione-S-transferase (GST) and metallothionein (MT) activity was measured in the fourth-instar chironomid larvae, Chironomus javanus, Kieffer, after either 48-hr or 96-hr exposure to organophosphorus insecticide, chlorpyrifos (0.01- 0.25 mg kg(-1)) or heavy metal cadmium (0.1-25 mg kg(-1)). Exposure to chlorpyrifos (0.01 mg kg(-1)) at 48 and 96 hr significantly of AChE activity (64.2%-85.9%) and induced GST activity (33.9-63.8%) when compared with control (P < 0.05). Moreover, exposure to cadmium (0.1 mg kg(-1)) at 48 and 96 hr also showed significant increas GST activity (11.7-40%) and MT level (9.0%-70.5%) when compared with control (P < 0.05). The results indicated the impact of enzyme activity on chlorpyrifos and cadmium contamination. Activity of AChE, GST and MT could serve as potential biomarkers for assessment and biomonitoring the effects of insecticide and heavy metal contamination in tropical aquatic ecosystems. PMID:26688973

  11. Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

    2016-01-01

    Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. PMID:26494252

  12. Activity Assay of Glutathione S-Transferase (GSTs) Enzyme as a Diagnostic Biomarker for Liver Hydatid Cyst in Vitro

    PubMed Central

    MOATAMEDI POUR, Lila; FARAHNAK, Ali; MOLAEI RAD, Mohamadbagher; GOLMOHAMADI, Taghi; ESHRAGHIAN, Mohamadreza

    2014-01-01

    Abstract Background The aim of this study was to detect the Glutathione S-Transferase(GST) enzyme activity of healthy / cystic liver as a diagnostic biomarker for hydatidosis. In order to compare with liver tissue, the level of the GSTs enzyme activity of parasite was also determined. Methods Parasites were collected from sheep liver tissue with hydatid cysts at a local abattoir and washed with PBS buffer. Collected parasites and liver tissues were sonicated or homogenized respectively. Extract solution samples were centrifuged and stored at - 20°C. GST enzyme activities were measured in the extract of parasite and liver tissue samples (healthy and infected livers). Protein amounts and protein bands were detected using Bradford and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods respectively. To determine significant difference between two groups, two-sample t-test was performed. Results GST specific activities of healthy / infected livers and parasites were estimated 304, 1297 and 146 U/ml/mg respectively. Significant higher GST specific activities in cystic liver than healthy liver was observed (P <0.05). T-test analysis showed GST activity of parasite was lower than healthy liver tissue. SDS-PAGE showed GST protein bands with 24 kDa in parasite samples and25 kDa in liver tissues. Conclusion GST activity incystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other hydatid disease parameters. PMID:25909067

  13. Modification of the association between maternal smoke exposure and congenital heart defects by polymorphisms in glutathione S-transferase genes

    PubMed Central

    Li, Xiaohong; Liu, Zhen; Deng, Ying; Li, Shengli; Mu, Dezhi; Tian, Xiaoxian; Lin, Yuan; Yang, Jiaxiang; Li, Jun; Li, Nana; Wang, Yanping; Chen, Xinlin; Deng, Kui; Zhu, Jun

    2015-01-01

    Congenital heart defects (CHDs) arise through various combinations of genetic and environmental factors. Our study explores how polymorphisms in the glutathione S-transferase (GST) genes affect the association between cigarette smoke exposure and CHDs. We analysed 299 mothers of children with CHDs and 284 mothers of children without any abnormalities who were recruited from six hospitals. The hair nicotine concentration (HNC) was used to quantify maternal smoke exposure, and the maternal GSTT1, and GSTM1 and GSTP1 genes were sequenced. We found a trend of higher adjusted odds ratios with higher maternal HNC levels, suggesting a dose-response relationship between maternal smoke exposure and CHDs. The lowest HNC range associated with an increased risk of CHDs was 0.213–0.319 ng/mg among the mothers with functional deletions of GSTM1 or GSTT1and 0.319–0.573 ng/mg among the mothers with normal copies of GSTM1 and GSTT1. In addition, the adjusted odds ratio for an HNC of >0.573 ng/mg was 38.53 among the mothers with the GSTP1 AG or GG genotype, which was 7.76 (χ2 = 6.702, p = 0.010) times greater than the AOR in the mothers with GSTP1 AA genotype. Our study suggests that polymorphisms of maternal GST genes may modify the association of maternal smoke exposure with CHDs. PMID:26456689

  14. Problematic detoxification of estrogen quinones by NAD(P)H-dependent quinone oxidoreductase and glutathione-S-transferase.

    PubMed

    Chandrasena, R Esala P; Edirisinghe, Praneeth D; Bolton, Judy L; Thatcher, Gregory R J

    2008-07-01

    Estrogen exposure through early menarche, late menopause, and hormone replacement therapy increases the risk factor for hormone-dependent cancers. Although the molecular mechanisms are not completely established, DNA damage by quinone electrophilic reactive intermediates, derived from estrogen oxidative metabolism, is strongly implicated. A current hypothesis has 4-hydroxyestrone-o-quinone (4-OQE) acting as the proximal estrogen carcinogen, forming depurinating DNA adducts via Michael addition. One aspect of this hypothesis posits a key role for NAD(P)H-dependent quinone oxidoreductase (NQO1) in the reduction of 4-OQE and protection against estrogen carcinogenesis, despite two reports that 4-OQE is not a substrate for NQO1. 4-OQE is rapidly and efficiently trapped by GSH, allowing measurement of NADPH-dependent reduction of 4-OQE in the presence and absence of NQO1. 4-OQE was observed to be a substrate for NQO1, but the acceleration of NADPH-dependent reduction by NQO1 over the nonenzymic reaction is less than 10-fold and at more relevant nanomolar concentrations of substrate is less than 2-fold. An alternative detoxifying enzyme, glutathione-S-transferase, was observed to be a target for 4-OQE, rapidly undergoing covalent modification. These results indicate that a key role for NQO1 and GST in direct detoxification of 4-hydroxy-estrogen quinones is problematic. PMID:18588320

  15. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  16. Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4-hydroxynon-2-enal

    PubMed Central

    Ayyadevara, Srinivas; Dandapat, Abhijit; Singh, Sharda P.; Siegel, Eric R.; Shmookler Reis, Robert J.; Zimniak, Ludwika; Zimniak, Piotr

    2007-01-01

    The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue-specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an "aging effector" via the formation of 4-HNE-protein adducts, and a resulting change in protein function. PMID:17157356

  17. Characterization of glutathione S-transferases from Sus scrofa, Cydia pomonella and Triticum aestivum: their responses to cantharidin.

    PubMed

    Yang, Xue-Qing; Zhang, Ya-Lin

    2015-02-01

    Glutathione S-transferases (GSTs) play a key role in detoxification of xenobiotics in organisms. However, their other functions, especially response to the natural toxin cantharidin produced by beetles in the Meloidae and Oedemeridae families, are less known. We obtained GST cDNAs from three sources: Cydia pomonella (CpGSTd1), Sus scrofa (SsGSTα1), and Triticum aestivum (TaGSTf3). The predicted molecular mass is 24.19, 25.28 and 24.49 kDa, respectively. These proteins contain typical N-terminal and C-terminal domains. Recombinant GSTs were heterologously expressed in Escherichia coli as soluble fusion proteins. Their optimal activities are exhibited at pH 7.0-7.5 at 30 °C. Activity of CpGSTd1 is strongly inhibited by cantharidin and cantharidic acid, but is only slightly suppressed by the demethylated analog of cantharidin and cantharidic acid. Enzymatic assays revealed that cantharidin has no effect on SsGSTα1 activity, while it significantly stimulates TaGSTf3 activity, with an EC50 value of 0.3852 mM. Activities of these proteins are potently inhibited by the known GST competitive inhibitor: S-hexylglutathione (GTX). Our results suggest that these GSTs from different sources share similar structural and biochemical characteristics. Our results also suggest that CpGSTd1 might act as a binding protein with cantharidin and its analogs. PMID:25640718

  18. Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione-S-transferase π

    PubMed Central

    McMillan, David H.; van der Velden, Jos L.J.; Lahue, Karolyn G.; Qian, Xi; Schneider, Robert W.; Iberg, Martina S.; Nolin, James D.; Abdalla, Sarah; Casey, Dylan T.; Tew, Kenneth D.; Townsend, Danyelle M.; Henderson, Colin J.; Wolf, C. Roland; Butnor, Kelly J.; Taatjes, Douglas J.; Budd, Ralph C.; Irvin, Charles G.; van der Vliet, Albert; Flemer, Stevenson; Anathy, Vikas; Janssen-Heininger, Yvonne M.W.

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-S-transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFβ-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp−/− mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFβ-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF. PMID:27358914

  19. Crystal structure of farnesyl protein transferase complexed with a CaaX peptide and farnesyl diphosphate analogue.

    PubMed

    Strickland, C L; Windsor, W T; Syto, R; Wang, L; Bond, R; Wu, Z; Schwartz, J; Le, H V; Beese, L S; Weber, P C

    1998-11-24

    The crystallographic structure of acetyl-Cys-Val-Ile-selenoMet-COOH and alpha-hydroxyfarnesylphosphonic acid (alphaHFP) complexed with rat farnesyl protein transferase (FPT) (space group P61, a = b = 174. 13 A, c = 69.71 A, alpha = beta = 90 degrees, gamma = 120 degrees, Rfactor = 21.8%, Rfree = 29.2%, 2.5 A resolution) is reported. In the ternary complex, the bound substrates are within van der Waals contact of each other and the FPT enzyme. alphaHFP binds in an extended conformation in the active-site cavity where positively charged side chains and solvent molecules interact with the phosphate moiety and aromatic side chains pack adjacent to the isoprenoid chain. The backbone of the bound CaaX peptide adopts an extended conformation, and the side chains interact with both FPT and alphaHFP. The cysteine sulfur of the bound peptide coordinates the active-site zinc. Overall, peptide binding and recognition appear to be dominated by side-chain interactions. Comparison of the structures of the ternary complex and unliganded FPT [Park, H., Boduluri, S., Moomaw, J., Casey, P., and Beese, L. (1997) Science 275, 1800-1804] shows that major rearrangements of several active site side chains occur upon substrate binding. PMID:9843427

  20. Vitamin E, glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured hepatocytes of rats treated with carcinogens.

    PubMed

    Ong, F B; Wan Ngah, W Z; Top, A G; Khalid, B A; Shamaan, N A

    1994-03-01

    1. The effects of alpha-tocopherol and gamma-tocotrienol on glutathione S-transferase (GST) and gamma-glutamyl transpeptidase (gamma-GT) activities in cultured hepatocytes prepared from rats treated with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were investigated. 2. Both the alpha-tocopherol and gamma-tocotrienol treated hepatocytes showed significantly higher (P < 0.05) GST activities than untreated hepatocytes prepared from the carcinogen treated rats in the first 3 days of culture. Treatment with alpha-tocopherol and gamma-tocotrienol generally resulted in a tendency to increase the GST activities above that in the untreated hepatocytes. 3. Treatment with high doses (125-250 microM) of alpha-tocopherol and low doses (12.5-25 microM) of gamma-tocotrienol generally resulted in a significant reduction in gamma-GT activities at 1-3 days. gamma-GT activities are reduced as the dose of alpha-tocopherol and gamma-tocotrienol are increased. PMID:7910569

  1. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

    NASA Astrophysics Data System (ADS)

    Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

    2015-05-01

    Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

  2. Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic.

    PubMed

    Todorova, Tatina T; Kujumdzieva, Anna V; Vuilleumier, Stéphane

    2010-11-01

    The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2-2.5-fold upon addition of either arsenate (As(V)) or arsenite (As(III)). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3'-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation. PMID:20740275

  3. The Glutathione-S-Transferase, Cytochrome P450 and Carboxyl/Cholinesterase Gene Superfamilies in Predatory Mite Metaseiulus occidentalis.

    PubMed

    Wu, Ke; Hoy, Marjorie A

    2016-01-01

    Pesticide-resistant populations of the predatory mite Metaseiulus (= Typhlodromus or Galendromus) occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) have been used in the biological control of pest mites such as phytophagous Tetranychus urticae. However, the pesticide resistance mechanisms in M. occidentalis remain largely unknown. In other arthropods, members of the glutathione-S-transferase (GST), cytochrome P450 (CYP) and carboxyl/cholinesterase (CCE) gene superfamilies are involved in the diverse biological pathways such as the metabolism of xenobiotics (e.g. pesticides) in addition to hormonal and chemosensory processes. In the current study, we report the identification and initial characterization of 123 genes in the GST, CYP and CCE superfamilies in the recently sequenced M. occidentalis genome. The gene count represents a reduction of 35% compared to T. urticae. The distribution of genes in the GST and CCE superfamilies in M. occidentalis differs significantly from those of insects and resembles that of T. urticae. Specifically, we report the presence of the Mu class GSTs, and the J' and J" clade CCEs that, within the Arthropoda, appear unique to Acari. Interestingly, the majority of CCEs in the J' and J" clades contain a catalytic triad, suggesting that they are catalytically active. They likely represent two Acari-specific CCE clades that may participate in detoxification of xenobiotics. The current study of genes in these superfamilies provides preliminary insights into the potential molecular components that may be involved in pesticide metabolism as well as hormonal/chemosensory processes in the agriculturally important M. occidentalis. PMID:27467523

  4. TA-3037A, a new inhibitor of glutathione S-transferase, produced by actinomycetes. I. Production, isolation, physico-chemical properties and biological activities.

    PubMed

    Komagata, D; Sawa, T; Muraoka, Y; Imada, C; Okami, Y; Takeuchi, T

    1992-07-01

    TA-3037A, a new inhibitor of glutathione S-transferase was discovered in the fermentation broth of Streptomyces sp. TA-3037. It was purified by chromatography followed by solvent extraction and then isolated as yellow needles. TA-3037A has the molecular formula of C16H11NO4. It was competitive with the substrate, and the inhibition constant (Ki) was 4.9 microM. PMID:1517156

  5. Synthetic fragments of antigenic lipophosphoglycans from Leishmania major and Leishmania mexicana and their use for characterisation of the Leishmania elongating alpha-D-mannopyranosylphosphate transferase.

    PubMed

    Higson, Adrian P; Ross, Andrew J; Tsvetkov, Yury E; Routier, Françoise H; Sizova, Olga V; Ferguson, Michael A J; Nikolaev, Andrei V

    2005-03-18

    The phosphorylated branched heptasaccharides 7 and 8, the octasaccharide 9 and the phosphorylated trisaccharides 5 and 6, which are fragments of the phosphoglycan portion of the surface lipophosphoglycans from Leishmania mexicana (5) or L. major (6-9), were synthesised by using the glycosyl hydrogenphosphonate method for the preparation of phosphodiester bridges. The compounds were tested as acceptor substrates/putative inhibitors for the Leishmania elongating alpha-D-mannosylphosphate transferase. PMID:15685582

  6. Terminal deoxynucleotidyl transferase activity and cell surface antigens of two unique cell lines (NALM-1 and BALM-2) of human leukemic origin.

    PubMed

    Sahai Srivastava, B I; Minowada, J

    1977-08-15

    Two unique cell lines, NALM-1 and BALM-2 derived from lymphoblast-like cells of chronic myelogenous leukemia and rare B cell acute lymphoblastic leukemia patients, respectively, were compared with fresh parent cells from the patients and with a Philadelphia chromosome positive K-562 cell line previously established from a chronic myelogenous leukemia patient in blastic phase. NALM-1 resembled the parent cells in the presence of Philadelphia chromosome, non-T/non-B acute lymphoblastic leukemia specific antigens and lack of T or B cell markers, whereas BALB-2, like the parent cells, had two chromosome markers and bore kappa, delta and mu immunoglobulins. NALM-1 lacked Epstein-Barr virus genome, whereas BALM-2 showed the presence of Epstein-Barr virus genome. K-562 cells lacked all the antigen markers examined. All cells had high DNA polymerase alpha activity and low DNA polymerase gamma activity. NALM-1, like the parent cells and unlike K-562 cells, had high terminal deoxynucleotidyl transferase activity of about 200 mu/mg DNA, whereas BALM-2, like its parent cells, had terminal deoxynucleotidyl transferase activity of 1-2 mu/mg DNA (1 u = 1 nmole Mn++-dGTP/h on dA12-18 initiator). Terminal deoxynucleotidyl transferase was characterized by its chromatographic and sedimentation behavior, thermal sensitivity and specific inhibition by streptolydigin and terminal deoxynucleotidyl transferase antisera. These results indicate that NALM-1 and K-562 may represent different phenotypes of cells in CML blastic crisis. Moreover, NALM-1 and BALM-2 seem to have retained the characteristics of original leukemic cells from which they may have been derived. PMID:70413

  7. Does Occupational Exposure to Solvents and Pesticides in Association with Glutathione S-Transferase A1, M1, P1, and T1 Polymorphisms Increase the Risk of Bladder Cancer? The Belgrade Case-Control Study

    PubMed Central

    Savic-Radojevic, Ana R.; Bulat, Petar V.; Pljesa-Ercegovac, Marija S.; Dragicevic, Dejan P.; Djukic, Tatjana I.; Simic, Tatjana P.; Pekmezovic, Tatjana D.

    2014-01-01

    Objective We investigated the role of the glutathione S-transferase A1, M1, P1 and T1 gene polymorphisms and potential effect modification by occupational exposure to different chemicals in Serbian bladder cancer male patients. Patients and Methods A hospital-based case-control study of bladder cancer in men comprised 143 histologically confirmed cases and 114 age-matched male controls. Deletion polymorphism of glutathione S-transferase M1 and T1 was identified by polymerase chain reaction method. Single nucleotide polymorphism of glutathione S-transferase A1 and P1 was identified by restriction fragment length polymorphism method. As a measure of effect size, odds ratio (OR) with corresponding 95% confidence interval (95%CI) was calculated. Results The glutathione S-transferase A1, T1 and P1 genotypes did not contribute independently toward the risk of bladder cancer, while the glutathione S-transferase M1-null genotype was overrepresented among cases (OR = 2.1, 95% CI = 1.1–4.2, p = 0.032). The most pronounced effect regarding occupational exposure to solvents and glutathione S-transferase genotype on bladder cancer risk was observed for the low activity glutathione S-transferase A1 genotype (OR = 9.2, 95% CI = 2.4–34.7, p = 0.001). The glutathione S-transferase M1-null genotype also enhanced the risk of bladder cancer among subjects exposed to solvents (OR = 6,5, 95% CI = 2.1–19.7, p = 0.001). The risk of bladder cancer development was 5.3–fold elevated among glutathione S-transferase T1-active patients exposed to solvents in comparison with glutathione S-transferase T1-active unexposed patients (95% CI = 1.9–15.1, p = 0.002). Moreover, men with glutathione S-transferase T1-active genotype exposed to pesticides exhibited 4.5 times higher risk in comparison with unexposed glutathione S-transferase T1-active subjects (95% CI = 0.9–22.5, p = 0.067). Conclusion Null or low-activity genotypes of the

  8. WaaA of the hyperthermophilic bacterium Aquifex aeolicus is a monofunctional 3-deoxy-D-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.

    PubMed

    Mamat, Uwe; Schmidt, Helgo; Munoz, Eva; Lindner, Buko; Fukase, Koichi; Hanuszkiewicz, Anna; Wu, Jing; Meredith, Timothy C; Woodard, Ronald W; Hilgenfeld, Rolf; Mesters, Jeroen R; Holst, Otto

    2009-08-14

    The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate. PMID:19546212

  9. Glutathione Transferase as a Potential Marker for Gut Epithelial Injury versus the Protective Role of Breast Milk sIgA in Infants with Rota Virus Gastroenteritis

    PubMed Central

    Sherif, Lobna S.; Raouf, Randaa K. Abdel; Sayede, Rokaya M. El; Wakkadd, Amany S. El; Shoaib, Ashraf R.; Ali, Hanan M.; Refay, Amira S. El

    2015-01-01

    BACKGROUND: Secretory immunoglobulin A (SIgA) plays an important protective role in the recognition and clearance of enteric pathogens. AIM: This study was designed to assess if mucosal integrity “measured by secretory IgA (SIgA)” is a protective factor from more epithelial alteration “measured by glutathione transferase” in infants with Rota gastroenteritis and its relation to infants’ feeding pattern. PATIENTS AND METHODS: This study was conducted on 79 infants aged 6 months and less from those diagnosed as having gastroenteritis and admitted to Gastroenteritis Department in Abo El Rish Pediatric Hospital, Cairo University. Plasma glutathione s-transferases and Stool SIgA were measured using ELISA technique. Rota virus detection was done by Reverse transcriptase PCR. RESULTS: SIgA was found to be significantly positive in exclusive breast fed infants, Glutathione transferase was significantly more frequently positive in Rota positive cases than Rota negative cases by Reverse transcriptase PCR. A significant negative correlation between Glutathione transferase and Secretory IgA was found, (p < 0.05). CONCLUSION: Breast feeding should be encouraged and highly recommended in the first two years of life as it provides Secretory IgA to breast fed infants who in turn protect them against epithelial damage caused by Rota viral gastroenteritis.

  10. S-(4-bromo-2,3-dioxobutyl)glutathione: A new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase

    SciTech Connect

    Katusz, R.M.; Colman, R.F. )

    1991-11-26

    S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 {mu}M) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25C results in a time-dependent inactivation of the enzyme. The k{sub obs} exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 {mu}M. Modified enzyme, prepared by incubating glutathione S-transferase with ({sup 3}H)S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH{sub 4}, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified. These results suggest that S-BDB-G functions as an affinity label at or near the active site of glutathione S-transferase and that modification of one site per enzyme subunit causes inactivation. It is proposed that the new compound, S-(4-bromo-2,3-dioxobutyl)glutathione, may have general applicability as an affinity label of other enzymes with glutathione binding sites.

  11. WaaA of the Hyperthermophilic Bacterium Aquifex aeolicus Is a Monofunctional 3-Deoxy-d-manno-oct-2-ulosonic Acid Transferase Involved in Lipopolysaccharide Biosynthesis*

    PubMed Central

    Mamat, Uwe; Schmidt, Helgo; Munoz, Eva; Lindner, Buko; Fukase, Koichi; Hanuszkiewicz, Anna; Wu, Jing; Meredith, Timothy C.; Woodard, Ronald W.; Hilgenfeld, Rolf; Mesters, Jeroen R.; Holst, Otto

    2009-01-01

    The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli ΔwaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate. PMID:19546212

  12. A member of the maize isopentenyl transferase gene family, Zea mays isopentenyl transferase 2 (ZmIPT2), encodes a cytokinin biosynthetic enzyme expressed during kernel development. Cytokinin biosynthesis in maize.

    PubMed

    Brugière, Norbert; Humbert, Sabrina; Rizzo, Nancy; Bohn, Jennifer; Habben, Jeffrey E

    2008-06-01

    Cytokinins (CKs) are plant hormones that regulate a large number of processes associated with plant growth and development such as induction of stomata opening, delayed senescence, suppression of auxin-induced apical dominance, signaling of nitrogen availability, differentiation of plastids and control of sink strength. In maize, CKs are thought to play an important role in establishing seed size and increasing seed set under normal and unfavorable environmental conditions therefore influencing yield. In recent years, the discovery of isopentenyl transferase (IPT) genes in plants has shed light on the CK biosynthesis pathway in plants. In an effort to increase our understanding of the role played by CKs in maize development and sink-strength, we identified several putative IPT genes using a bioinformatics approach. We focused our attention on one gene in particular, ZmIPT2, because of its strong expression in developing kernels. The expression of the gene and its product overlays the change in CK levels in developing kernels suggesting a major role in CK biosynthesis for kernel development. We demonstrate that at 8-10 days after pollination (DAP) the endosperm and especially the basal transfer cell layer (BETL) is a major site of ZmIPT2 expression, and that this expression persists in the BETL and the developing embryo into later kernel development stages. We show that ectopic expression of ZmIPT2 in calli and in planta created phenotypes consistent with CK overproduction. We also show that ZmIPT2 preferentially uses ADP and ATP over AMP as the substrates for dimethylallyl diphosphate (DMAPP) IPT activity. The expression pattern of ZmIPT2 in the BETL, endosperm and embryo during kernel development will be discussed with an emphasis on the suggested role of CKs in determining sink-strength and grain production in crop plants. PMID:18311542

  13. Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme.

    PubMed Central

    Hao, X Y; Widersten, M; Ridderström, M; Hellman, U; Mannervik, B

    1994-01-01

    Qualitative and quantitative analyses of glutathione, glutathione transferases (GSTs) and other glutathione-linked enzymes in HeLa cells have been made in order to study their significance in cellular resistance to electrophilic cytotoxic agents. The cytosolic concentrations of three GSTs, GST M1-1 (53 +/- 9 ng/mg of cytosolic protein), GST P1-1 (11 +/- 3 ng/mg) and GST A1-1 (1.1 +/- 0.4 ng/mg) were quantified by isoenzyme-specific enzyme-linked immunoassays. Electrophoretic analysis and immunoblotting demonstrated another component, GST M3-3, which was identified by amino acid sequence analysis. GST M3-3 was quantified (1550 +/- 250 ng/mg) by slot-blot immunoanalysis and was the most abundant GST in HeLa cells. An additional cytosolic 13 kDa protein with high affinity for immobilized glutathione or S-hexyglutathione was found to be identical with a macrophage migration-inhibitory factor, previously identified as a lymphokine. Cells grown in roller bottles (HR) rather than in ordinary culture flasks contain a significantly lower concentration of all the GSTs and were found to be more sensitive to the cytostatic agents doxorubicin (2.3-fold), cisplatin (1.7-fold) and melphalan (1.4-fold). The cytosolic concentrations of glutathione reductase and glyoxalase I were also lower in HR cells, whereas the total glutathione concentration was unchanged and the glutathione peroxidase activity was increased. The results indicate that GSTs contribute to the cellular resistance phenotype. Images Figure 1 Figure 2 Figure 4 PMID:8280111

  14. Differential Activation of Diverse Glutathione Transferases of Clonorchis sinensis in Response to the Host Bile and Oxidative Stressors

    PubMed Central

    Bae, Young-An; Ahn, Do-Whan; Lee, Eung-Goo; Kim, Seon-Hee; Cai, Guo-Bin; Kang, Insug; Sohn, Woon-Mok; Kong, Yoon

    2013-01-01

    Background Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other. Methodology/Principal Findings We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular

  15. Polymorphisms of glutathione S-transferase and methylenetetrahydrofolate reductase genes in Moldavian patients with ulcerative colitis: Genotype-phenotype correlation

    PubMed Central

    Varzari, Alexander; Deyneko, Igor V.; Tudor, Elena; Turcan, Svetlana

    2015-01-01

    Background Glutathione S-transferases (GSTM1, GSTT1, and GSTP1) and methylenetetrahydrofolate reductase (MTHFR) are important enzymes for protection against oxidative stress. In addition, MTHFR has an essential role in DNA synthesis, repair, and methylation. Their polymorphisms have been implicated in the pathogenesis of ulcerative colitis (UC). The aim of the present study was to investigate the role of selected polymorphisms in these genes in the development of UC in the Moldavian population. Methods In a case-control study including 128 UC patients and 136 healthy individuals, GSTM1 and GSTT1 genotypes (polymorphic deletions) were determined using multiplex polymerase chain reaction (PCR). The GSTP1 rs1695 (Ile105Val), MTHFR rs1801133 (C677T), and MTHFR rs1801131 (A1298C) polymorphisms were studied with restriction fragment length polymorphism (RFLP) analysis. Genotype–phenotype correlations were examined using logistic regression analysis. Results None of the genotypes, either alone or in combination, showed a strong association with UC. The case-only sub-phenotypic association analysis showed an association of the MTHFR rs1801133 polymorphism with the extent of UC under co-dominant (p corrected = 0.040) and recessive (p corrected = 0.020; OR = 0.15; CI = 0.04–0.63) genetic models. Also, an association between the MTHFR rs1801131 polymorphism and the severity of UC was reported for the over-dominant model (p corrected = 0.023; coefficient = 0.32; 95% CI = 0.10–0.54). Conclusion The GST and MTHFR genotypes do not seem to be a relevant risk factor for UC in our sample. There was, however, evidence that variants in MTHFR may influence the clinical features in UC patients. Additional larger studies investigating the relationship between GST and MTHFR polymorphisms and UC are required. PMID:26862484

  16. Decreased glutathione transferase levels in rd1/rd1 mouse retina: replenishment protects photoreceptors in retinal explants.

    PubMed

    Ahuja, P; Caffé, A R; Ahuja, S; Ekström, P; van Veen, T

    2005-01-01

    Currently much attention is focused on glutathione S transferase (GST)-induced suppression of apoptosis. The objective of our studies was therefore to see if GST isoenzymes rescue photoreceptors in retinal explants from rd1/rd1 mice, in which photoreceptors degenerate rapidly. Eyes from C3H rd1/rd1 and +/+ mice were collected at various time points between postnatal day (PN) 2 and PN28. Localization and content of alpha-GST and mu-GST was investigated by immunofluorescence and semi-quantitative Western blot analysis, respectively. In addition, PN2 and PN7 retinal explants were cultured till PN28, during which they were treated with 10 ng/ml alpha-GST or mu-GST. The spatiotemporal expression of both GST isoforms was closely similar: early presence in ganglion cell layer after which staining became restricted to Muller cells (particularly in the endfeet) and horizontal cell fibers in both rd1/rd1 and +/+. Doublets of alpha-GST and mu-GST were detected by Western blot analysis. Densitometry of these bands indicated steady reduction of alpha-GST content in rd1/rd1 retina starting from the second postnatal week. When alpha-GST and mu-GST were added exogenously to rd1/rd1 explants, photoreceptor rescue was produced that was more prominent in PN2 than in PN7 explants and more effective by alpha-GST than mu-GST. We propose that alpha-GST neuroprotection is mediated by reduction of tissue oxidative stress. PMID:15749346

  17. Directed evolution of Tau class glutathione transferases reveals a site that regulates catalytic efficiency and masks co-operativity.

    PubMed

    Axarli, Irine; Muleta, Abdi W; Vlachakis, Dimitrios; Kossida, Sophia; Kotzia, Georgia; Maltezos, Anastasios; Dhavala, Prathusha; Papageorgiou, Anastassios C; Labrou, Nikolaos E

    2016-03-01

    A library of Tau class GSTs (glutathione transferases) was constructed by DNA shuffling using the DNA encoding the Glycine max GSTs GmGSTU2-2, GmGSTU4-4 and GmGSTU10-10. The parental GSTs are >88% identical at the sequence level; however, their specificity varies towards different substrates. The DNA library contained chimaeric structures of alternated segments of the parental sequences and point mutations. Chimaeric GST sequences were expressed in Escherichia coli and their enzymatic activities towards CDNB (1-chloro-2,4-dinitrobenzene) and the herbicide fluorodifen (4-nitrophenyl α,α,α-trifluoro-2-nitro-p-tolyl ether) were determined. A chimaeric clone (Sh14) with enhanced CDNB- and fluorodifen-detoxifying activities, and unusual co-operative kinetics towards CDNB and fluorodifen, but not towards GSH, was identified. The structure of Sh14 was determined at 1.75 Å (1 Å=0.1 nm) resolution in complex with S-(p-nitrobenzyl)-glutathione. Analysis of the Sh14 structure showed that a W114C point mutation is responsible for the altered kinetic properties. This was confirmed by the kinetic properties of the Sh14 C114W mutant. It is suggested that the replacement of the bulky tryptophan residue by a smaller amino acid (cysteine) results in conformational changes of the active-site cavity, leading to enhanced catalytic activity of Sh14. Moreover, the structural changes allow the strengthening of the two salt bridges between Glu(66) and Lys(104) at the dimer interface that triggers an allosteric effect and the communication between the hydrophobic sites. PMID:26637269

  18. Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals

    PubMed Central

    Goodrich, Jaclyn M.; Basu, Niladri

    2012-01-01

    Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from E. coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p<0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean ±SEM: Ile105 Ala114 Km= 0.33±0.07 mM vs. Val105 Ala114 Km=1.15±0.07 mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic- HgCl2, methylmercury- MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl2 (IC50 range: 24.1–172 μM), MeHg (93.9–480 μM), and selenium (43.7–62.8 μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl2 and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species. PMID:22401947

  19. Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam

    SciTech Connect

    Agusa, Tetsuro; Iwata, Hisato; Fujihara, Junko; Kunito, Takashi; Takeshita, Haruo; Tu Binh Minh; Pham Thi Kim Trang; Pham Hung Viet; Tanabe, Shinsuke

    2010-02-01

    To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST omega1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST omega2 (GSTO2) Asn142Asp, GST pi1 (GSTP1) Ile105Val, GST mu1 (GSTM1) wild/null, and GST theta1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As{sup V} than the wild homo type. Higher percentage of DMA{sup V} in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As{sup V} to As{sup III}. Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population.

  20. Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus

    PubMed Central

    Zhang, Yuanwei; Zheng, Qingqing; Sun, Congcong; Song, Jinxing; Gao, Lina; Zhang, Shizhu; Muñoz, Alberto; Read, Nick D.; Lu, Ling

    2016-01-01

    Finely tuned changes in cytosolic free calcium ([Ca2+]c) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS). The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs), a putative proton V-type proton ATPase (Vma5 homolog) and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress. PMID:27058039

  1. Effects of high-intensity intermittent training on carnitine palmitoyl transferase activity in the gastrocnemius muscle of rats.

    PubMed

    Carnevali Jr, L C; Eder, R; Lira, F S; Lima, W P; Gonçalves, D C; Zanchi, N E; Nicastro, H; Lavoie, J M; Seelaender, M C L

    2012-08-01

    We examined the capacity of high-intensity intermittent training (HI-IT) to facilitate the delivery of lipids to enzymes responsible for oxidation, a task performed by the carnitine palmitoyl transferase (CPT) system in the rat gastrocnemius muscle. Male adult Wistar rats (160-250 g) were randomly distributed into 3 groups: sedentary (Sed, N = 5), HI-IT (N = 10), and moderate-intensity continuous training (MI-CT, N = 10). The trained groups were exercised for 8 weeks with a 10% (HI-IT) and a 5% (MI-CT) overload. The HI-IT group presented 11.8% decreased weight gain compared to the Sed group. The maximal activities of CPT-I, CPT-II, and citrate synthase were all increased in the HI-IT group compared to the Sed group (P < 0.01), as also was gene expression, measured by RT-PCR, of fatty acid binding protein (FABP; P < 0.01) and lipoprotein lipase (LPL; P < 0.05). Lactate dehydrogenase also presented a higher maximal activity (nmol·min(-1)·mg protein(-1)) in HI-IT (around 83%). We suggest that 8 weeks of HI-IT enhance mitochondrial lipid transport capacity thus facilitating the oxidation process in the gastrocnemius muscle. This adaptation may also be associated with the decrease in weight gain observed in the animals and was concomitant to a higher gene expression of both FABP and LPL in HI-IT, suggesting that intermittent exercise is a "time-efficient" strategy inducing metabolic adaptation. PMID:22735180

  2. Effects of high-intensity intermittent training on carnitine palmitoyl transferase activity in the gastrocnemius muscle of rats

    PubMed Central

    Carnevali, L.C.; Eder, R.; Lira, F.S.; Lima, W.P.; Gonçalves, D.C.; Zanchi, N.E.; Nicastro, H.; Lavoie, J.M.; Seelaender, M.C.L.

    2012-01-01

    We examined the capacity of high-intensity intermittent training (HI-IT) to facilitate the delivery of lipids to enzymes responsible for oxidation, a task performed by the carnitine palmitoyl transferase (CPT) system in the rat gastrocnemius muscle. Male adult Wistar rats (160-250 g) were randomly distributed into 3 groups: sedentary (Sed, N = 5), HI-IT (N = 10), and moderate-intensity continuous training (MI-CT, N = 10). The trained groups were exercised for 8 weeks with a 10% (HI-IT) and a 5% (MI-CT) overload. The HI-IT group presented 11.8% decreased weight gain compared to the Sed group. The maximal activities of CPT-I, CPT-II, and citrate synthase were all increased in the HI-IT group compared to the Sed group (P < 0.01), as also was gene expression, measured by RT-PCR, of fatty acid binding protein (FABP; P < 0.01) and lipoprotein lipase (LPL; P < 0.05). Lactate dehydrogenase also presented a higher maximal activity (nmol·min−1·mg protein−1) in HI-IT (around 83%). We suggest that 8 weeks of HI-IT enhance mitochondrial lipid transport capacity thus facilitating the oxidation process in the gastrocnemius muscle. This adaptation may also be associated with the decrease in weight gain observed in the animals and was concomitant to a higher gene expression of both FABP and LPL in HI-IT, suggesting that intermittent exercise is a “time-efficient” strategy inducing metabolic adaptation. PMID:22735180

  3. Expression level and DNA methylation status of Glutathione-S-transferase genes in normal murine prostate and TRAMP tumors

    PubMed Central

    Mavis, Cory K.; Kinney, Shannon R. Morey; Foster, Barbara A.; Karpf, Adam R.

    2010-01-01

    BACKGROUND Glutathione-S-transferase (Gst) genes are down-regulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors. METHODS Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n=15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5’ CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured. RESULTS Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells. CONCLUSIONS Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression. PMID:19444856

  4. Novel function of glutathione transferase in rat liver mitochondrial membrane: Role for cytochrome c release from mitochondria

    SciTech Connect

    Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko

    2008-10-01

    Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore.

  5. The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

    PubMed Central

    Karpusas, Michael; Axarli, Irine; Chiniadis, Lykourgos; Papakyriakou, Athanasios; Bethanis, Kostas; Scopelitou, Katholiki; Clonis, Yannis D.; Labrou, Nikolaos E.

    2013-01-01

    Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated. PMID:23460799

  6. Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases.

    PubMed

    Jaffee, Marcie B; Imperiali, Barbara

    2011-09-01

    The central enzyme in N-linked glycosylation is the oligosaccharyl transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology to the Stt3 subunit of the complex multimeric yeast OTase. Sequence identity between PglB and Stt3 is low (17.9%); however, both have a similar predicted architecture and contain the conserved WWDxG motif. To investigate the relationship between PglB and other Stt3 proteins, sequence analysis was performed using 28 homologues from evolutionarily distant organisms. Since detection of small conserved motifs within large membrane-associated proteins is complicated by divergent sequences surrounding the motifs, we developed a program to parse sequences according to predicted topology and then analyze topologically related regions. This approach identified three conserved motifs that served as the basis for subsequent mutagenesis and functional studies. This work reveals that several inter-transmembrane loop regions of PglB/Stt3 contain strictly conserved motifs that are essential for PglB function. The recent publication of a 3.4 Å resolution structure of full-length C. lari OTase provides clear structural evidence that these loops play a fundamental role in catalysis [ Lizak , C. ; ( 2011 ) Nature 474 , 350 - 355 ]. The current study provides biochemical support for the role of the inter-transmembrane domain loops in OTase catalysis and demonstrates the utility of combining topology prediction and sequence analysis for exposing buried pockets of homology in large membrane proteins. The described approach allowed detection of the catalytic motifs prior to availability of structural data and reveals additional catalytically relevant residues that are not predicted by structural data alone. PMID:21812456

  7. Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress.

    PubMed Central

    Favaloro, B; Tamburro, A; Trofino, M A; Bologna, L; Rotilio, D; Heipieper, H J

    2000-01-01

    The gluthathione S-transferase gene of the atrazine-degrading bacterium Ochrobactrum anthropi (OaGST) encodes a single-subunit polypeptide of 201 amino acid residues (Favaloro et al. 1998, Biochem. J. 335, 573-579). RNA blot analysis showed that the gene is transcribed into an mRNA of about 800 nucleotides, indicating a monocistronic transcription of the OaGST gene. The modulation of OaGST in this bacterium, in the presence of different stimulants, was investigated. The level of expression of OaGST was detected both by measuring the mRNA level and by immunoblotting experiments. OaGST is a constitutive enzyme which is also inducible by several stimulants. In fact, atrazine caused an increase in the expression of OaGST even at concentrations which had no effect on growth rates of the bacteria. Moreover, the presence of other aromatic substrates of this bacterium, such as phenol and chlorophenols, leads to a marked enhancement in OaGST expression. In this case, the expression of OaGST was related to growth inhibition and membrane damage caused by these hydrophobic compounds, and to the adaptive responses of the cell membranes. On the other hand, toluene and xylene, two aromatic compounds not degradable by this bacterium, did not induce the OaGST expression. The same was observed for other stress conditions such as low pH, heat shock, hydrogen peroxide, osmotic stress, starvation, the presence of aliphatic alcohols or heavy metals. These results suggest a co-regulation of the OaGST gene by the catabolic pathways of phenols and chlorophenols in this bacterium. Therefore, OaGST could function as a detoxifying agent within the catabolism of these xenobiotics. PMID:10677378

  8. Garlic organosulfur compounds upregulate the expression of the pi class of glutathione S-transferase in rat primary hepatocytes.

    PubMed

    Tsai, Chia-Wen; Yang, Jaw-Ji; Chen, Haw-Wen; Sheen, Lee-Yan; Lii, Chong-Kuei

    2005-11-01

    The chemopreventive property of garlic is related in part to its induction of phase II detoxification enzymes. In the present study, we investigated the modulatory effect of 3 garlic organosulfur compounds, i.e., diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), which differ in their number of sulfur atoms, on the gene expression of the pi class of glutathione S-transferase (GSTP). Hepatocytes isolated from male Sprague-Dawley rats were cultured with 50-200 micromol/L of DAS, DADS, or DATS for 24 h. DADS and DATS increased GST activity toward ethacrynic acid by 40 and 66%, respectively (P < 0.05). Moreover, both garlic allyl sulfides dose dependently induced GSTP mRNA and protein expression. DATS increased the protein level more than DADS (P < 0.05). In contrast, DAS did not affect the activity or the protein or mRNA levels of this phase II drug-metabolizing enzyme. In Clone 9 liver cells, the pTA-luciferase reporter assay showed that luciferase activity in DADS- and DATS-treated cells was 2.8- and 3.9-fold higher than that in control cells, respectively (P < 0.05). Again, luciferase activity was not affected by treatment with DAS. Deletion of -2.7 to -2.6 kb in the GSTP promoter region, which contains the GSTP enhancer (GPE) I element, abolished the upregulation of GSTP transcription by DADS and DATS. Deletion of GPE II, however, did not affect the induction of reporter activity. In conclusion, the effectiveness of 3 garlic allyl sulfides on GSTP expression was related to the number of sulfur atoms in the molecules, and GPE I was responsible for this upregulation. PMID:16251611

  9. Glutathione S-Transferase (GST) Gene Diversity in the Crustacean Calanus finmarchicus – Contributors to Cellular Detoxification

    PubMed Central

    Roncalli, Vittoria; Cieslak, Matthew C.; Passamaneck, Yale; Christie, Andrew E.; Lenz, Petra H.

    2015-01-01

    Detoxification is a fundamental cellular stress defense mechanism, which allows an organism to survive or even thrive in the presence of environmental toxins and/or pollutants. The glutathione S-transferase (GST) superfamily is a set of enzymes involved in the detoxification process. This highly diverse protein superfamily is characterized by multiple gene duplications, with over 40 GST genes reported in some insects. However, less is known about the GST superfamily in marine organisms, including crustaceans. The availability of two de novo transcriptomes for the copepod, Calanus finmarchicus, provided an opportunity for an in depth study of the GST superfamily in a marine crustacean. The transcriptomes were searched for putative GST-encoding transcripts using known GST proteins from three arthropods as queries. The identified transcripts were then translated into proteins, analyzed for structural domains, and annotated using reciprocal BLAST analysis. Mining the two transcriptomes yielded a total of 41 predicted GST proteins belonging to the cytosolic, mitochondrial or microsomal classes. Phylogenetic analysis of the cytosolic GSTs validated their annotation into six different subclasses. The predicted proteins are likely to represent the products of distinct genes, suggesting that the diversity of GSTs in C. finmarchicus exceeds or rivals that described for insects. Analysis of relative gene expression in different developmental stages indicated low levels of GST expression in embryos, and relatively high expression in late copepodites and adult females for several cytosolic GSTs. A diverse diet and complex life history are factors that might be driving the multiplicity of GSTs in C. finmarchicus, as this copepod is commonly exposed to a variety of natural toxins. Hence, diversity in detoxification pathway proteins may well be key to their survival. PMID:25945801

  10. Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

    SciTech Connect

    Liu, Xiyuan; An, Byoung Ha; Kim, Min Jung; Park, Jong Hoon; Kang, Young Sook; Chang, Minsun

    2014-09-26

    Highlights: • GSTP induces the classical ERα signaling event. • The functional GSTP is a prerequisite for GSTP-induced ERα transcription activity. • The expression of RIP140, a transcription cofactor, was inhibited by GSTP protein. • We propose the novel non-enzymatic role of GSTP. - Abstract: Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.

  11. Tributyltin induces oxidative stress and neuronal injury by inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures.

    PubMed

    Ishihara, Yasuhiro; Kawami, Tomohito; Ishida, Atsuhiko; Yamazaki, Takeshi

    2012-06-01

    Tributyltin (TBT) has been used as a heat stabilizer, agricultural pesticide and antifouling agents on ships, boats and fish-farming nets; however, the neurotoxicity of TBT has recently become a concern. TBT is suggested to stimulate the generation of reactive oxygen species (ROS) inside cells. The aim of this study was to determine the mechanism of neuronal oxidative injury induced by TBT using rat organotypic hippocampal slice cultures. The treatment of rat hippocampal slices with TBT induced ROS production, lipid peroxidation and cell death. Pretreatment with antioxidants such as superoxide dismutase, catalase or trolox, suppressed the above phenomena induced by TBT, indicating that TBT elicits oxidative stress in hippocampal slices, which causes neuronal cell death. TBT dose-dependently inhibited glutathione S-transferase (GST), but not glutathione peroxidase or glutathione reductase in the cytosol of rat hippocampus. The treatment of hippocampal slices with TBT decreased the GST activity. Pretreatment with reduced glutathione attenuated the reduction of GST activity and cell death induced by TBT, indicating that the decrease in GST activity by TBT is involved in hippocampal cell death. When hippocampal slices were treated with sulforaphane, the expression and activity of GST were increased. Notably, TBT-induced oxidative stress and cell death were significantly suppressed by pretreatment with sulforaphane. These results indicate that GST inhibition could contribute, at least in part, to the neuronal cell death induced by TBT in hippocampal slices. This study is the first report to show the link between neuronal oxidative injury and the GST inhibition elicited by TBT. PMID:22449404

  12. Further evidence that rat liver microsomal glutathione transferase 1 is not a cellular protein target for S-nitrosylation.

    PubMed

    Shi, Qiang; Chen, Hai-Fei; Lou, Yi-Jia

    2006-09-25

    By adopting biotin switch method, we recently reported that liver microsomal glutathione transferase 1 (MGST1) might not be a protein target for S-nitrosylation in rat microsomes or in vivo. However, alternative analytic methods are needed to confirm this observation, as a single biotin switch method in judging specific protein S-nitrosylation in biological samples is increasingly recognized as insufficient, or even unreliable. Besides, only MGST1 localized on endoplasmic reticulum (ER), but not mitochondria which favors protein S-nitrosylation was examined in the previous report. Present study was therefore carried out to address these issues. Primary cultured hepatocytes were used. A physiological existing nitric oxide (NO) donor S-nitrosoglutathione (GSNO) was adopted to trigger protein S-nitrosylation. MGST1 was immunoprecipitated and its S-nitrosothiol content was measured by the NO probe 2,3-diaminonaphthalene. In parallel, S-nitrosylated proteins were immunoprecipitated by a monoclonal anti-S-nitrosocysteine antibody and probed with an anti-MGST1 antibody. In hepatocytes, neither ER nor mitochondria were found to contain S-nitrosylated MGST1 after GSNO treatment, showing that differently distributed MGST1 was consistently un-nitrosylable in the cellular environment. But under broken cell conditions, when samples were incubated directly with GSNO, MGST1 S-nitrosylation was indeed detectable in both the microsomal and mitochondrial proteins, indicating that previous failure in detecting MGST1 S-nitrosylation in microsomes is due to the limitations of biotin switch method. These results clearly, if not definitely, demonstrate that MGST1 is not a ready candidate for S-nitrosylation in the cellular content, despite its susceptibility to S-nitrosylation under broken cell conditions. PMID:16899233

  13. Evaluation of hepatic damage and local immune response in goats immunized with native glutathione S-transferase of Fasciola hepatica.

    PubMed

    Zafra, R; Pérez-Ecija, R A; Buffoni, L; Mendes, R E; Martínez-Moreno, A; Martínez-Moreno, F J; Galisteo, M E Martínez; Pérez, J

    2010-01-01

    Worm burden, hepatic damage and local cellular and humoral immune responses were assessed in goats immunized with glutathione-S-transferase and challenged with Fasciola hepatica. Infected but unimmunized and uninfected control groups were also studied. Hepatic damage was evaluated grossly and microscopically. Local immune response was evaluated by (1) microscopical examination of hepatic lymph nodes (HLNs); (2) analysis of the distribution of CD2(+), CD4(+), CD8(+), T-cell receptor gammadelta(+) lymphocytes and immunoglobulin (Ig) G(+) plasma cells; and (3) investigation of the distribution of cells expressing interleukin (IL)-4 and interferon (IFN)-gamma in the hepatic inflammatory infiltrates and HLNs. Immunized animals did not have significant reduction in fluke number, but there was significant (P<0.05) reduction of fluke size relative to the control groups. The lesions in the two infected groups were similar and consisted of fibrous perihepatitis and white tortuous tracts, mainly involving the left hepatic lobe. Microscopical lesions were similar in both infected groups and were typical of chronic fascioliosis. These included portal fibrosis, inflammatory infiltration with plasma cells, formation of lymphoid follicles, accumulation of haemosiderin-laden macrophages and granulomatous foci. Both infected groups had a marked local immune response characterized by infiltration of CD2(+), CD4(+) and CD8(+) T lymphocytes, and IgG(+) plasma cells in hepatic lesions and in HLNs. There was no expression of IL-4 or INF-gamma by cells in the hepatic inflammatory infiltrate, but expression of INF-gamma in HLNs was much lower than that of IL-4, suggesting an immune response dominated by T helper 2 cells. PMID:20185148

  14. Large-Scale Determination of Sequence, Structure, and Function Relationships in Cytosolic Glutathione Transferases across the Biosphere

    PubMed Central

    Mashiyama, Susan T.; Malabanan, M. Merced; Akiva, Eyal; Bhosle, Rahul; Branch, Megan C.; Hillerich, Brandan; Jagessar, Kevin; Kim, Jungwook; Patskovsky, Yury; Seidel, Ronald D.; Stead, Mark; Toro, Rafael; Vetting, Matthew W.; Almo, Steven C.; Armstrong, Richard N.; Babbitt, Patricia C.

    2014-01-01

    The cytosolic glutathione transferase (cytGST) superfamily comprises more than 13,000 nonredundant sequences found throughout the biosphere. Their key roles in metabolism and defense against oxidative damage have led to thousands of studies over several decades. Despite this attention, little is known about the physiological reactions they catalyze and most of the substrates used to assay cytGSTs are synthetic compounds. A deeper understanding of relationships across the superfamily could provide new clues about their functions. To establish a foundation for expanded classification of cytGSTs, we generated similarity-based subgroupings for the entire superfamily. Using the resulting sequence similarity networks, we chose targets that broadly covered unknown functions and report here experimental results confirming GST-like activity for 82 of them, along with 37 new 3D structures determined for 27 targets. These new data, along with experimentally known GST reactions and structures reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and reveal that the great majority of cytGSTs have not been experimentally characterized or annotated by canonical class. A mapping of taxonomic classes across the superfamily indicates that many taxa are represented in each subgroup and highlights challenges for classification of superfamily sequences into functionally relevant classes. Experimental determination of disulfide bond reductase activity in many diverse subgroups illustrate a theme common for many reaction types. Finally, sequence comparison between an enzyme that catalyzes a reductive dechlorination reaction relevant to bioremediation efforts with some of its closest homologs reveals differences among them likely to be associated with evolution of this unusual reaction

  15. The role of the inhibition of glutathione-S-transferase in the protective mechanisms of ischemic postconditioning.

    PubMed

    Balatonyi, Borbála; Gasz, Balázs; Kovács, Viktória; Lantos, János; Jancsó, Gábor; Marczin, Nándor; Rőth, Erzsébet

    2013-08-01

    The antioxidant glutathione-S-transferase (GST) is a crucial determinant of the development of ischaemic-reperfusion (I/R) injury, and plays a pivotal role in the regulation of the mitogen activated protein kinase (MAPK) pathways involved in stress response and apoptosis. The aim of this study was to investigate whether inhibition of GST can abolish the benefit of ischaemic postconditioning (IPoC). A neonatal rat cardiomyocyte cell culture was prepared and divided into 6 groups: (I) control group without treatment; (II) cells exposed to simulated I/R; (III) simulated I/R (sI/R) with IPoC; (IV) ethacrynic acid (EA) alone; (V) sI/R with EA; and (VI) sI/R and IPoC together with EA. Viability of the cells was measured by MTT assay, the quantity of apoptotic cells was assessed by flow cytometry following annexin V-FITC - propidium-iodide double staining. The activation of JNK, p38, ERK/p42-p44 MAPKs, and GSK-3β protein kinase was determined by flow-cytometric assay. GST inhibition markedly increased the apoptosis and decreased the cell viability despite IPoC. The protective effect of IPoC was lost in GST-inhibited groups for all MAPKs and GSK-3β. GST activity is required for the survival of cultured cardiomyocytes under stress conditions. GST inhibition was associated with differential activation of MAP and the protein kinases regulating these pathways in the process of ischaemic postconditioning. PMID:23888930

  16. Dual protective role for Glutathione S-transferase class pi against VCD-induced ovotoxicity in the rat ovary

    SciTech Connect

    Keating, Aileen F.; Sen, Nivedita; Sipes, I. Glenn; Hoyer, Patricia B.

    2010-09-01

    The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extra-ovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80 mg/kg/day; 15 days, i.p.). The present study investigated the pattern of ovarian GSTp expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium {+-} VCD (30 {mu}M) for 2-8 days. VCD increased ovarian GSTp mRNA (P < 0.05) relative to control on d4-d8; whereas GSTp protein was increased (P < 0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P = 0.09), while unbound JNK was decreased (P < 0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis.

  17. Association of Glutathione S-Transferase P1 (GSTP1) Polymorphism with Tourette Syndrome in Taiwanese Patients

    PubMed Central

    Shen, Che-Piao; Chou, I-Ching; Liu, Hsin-Ping; Lee, Cheng-Chun; Tsai, Yuhsin; Wu, Bor-Tsang; Hsu, Ban-Dar

    2014-01-01

    The etiology of Tourette syndrome (TS) is multifactorial. TS vulnerability may be associated with genetic and environmental factors. From the genetic point of view, TS is heterogeneous. Previous studies showed that some single-nucleotide polymorphisms (SNPs) of the glutathione-S-transferase P1 (GSTP1) gene can affect cellular proliferation and apoptotic activity and TS is a neurodevelopmental disorder. We guessed that there was a relationship between TS and genetic variants of the GSTP1 gene. Therefore, in this study, we aimed to test the hypothesis that GSTP1 SNPs were associated with TS. We performed a case–control study. One hundred twenty-one TS children and 105 normal children were included in the study. Polymerase chain reaction was used to identify the GSTP1 gene polymorphism at position rs6591256 (A/G, promoter polymorphism) in TS patients and normal children. The polymorphism at position rs6591256 in the GSTP1 gene revealed significant differences in the allele (p=0.0135) and genotype (p=0.0159) distributions between the TS patients and the control group. The A allele was present at a higher frequency than the G allele in the TS patients compared with the control group (odds ratio [OR]=1.91, 95% confidence interval [CI]: 1.14–3.21). The AA genotype was associated with susceptibility to TS with an OR of 2.38 for the AA versus AG genotype (95% CI: 1.29–4.41). These findings suggest that variants in the GSTP1 gene may play a role in susceptibility to TS. PMID:24205873

  18. Dietary isothiocyanates, glutathione S-transferase polymorphisms and colorectal cancer risk in the Singapore Chinese Health Study.

    PubMed

    Seow, Adeline; Yuan, Jian-Min; Sun, Can-Lan; Van Den Berg, David; Lee, Hin-Peng; Yu, Mimi C

    2002-12-01

    Dietary intake of cruciferous vegetables (Brassica spp.) has been inversely related to colorectal cancer risk, and this has been attributed to their high content of glucosinolate degradation products such as isothiocyanates (ITCs). These compounds act as anticarcinogens by inducing phase II conjugating enzymes, in particular glutathione S-transferases (GSTs). These enzymes also metabolize ITCs, such that the protective effect of cruciferous vegetables may predicate on GST genotype. The Singapore Chinese Health Study is a prospective investigation among 63 257 middle-aged men and women, who were enrolled between April 1993 and December 1998. In this nested case-control analysis, we compared 213 incident cases of colorectal cancer with 1194 controls. Information on dietary ITC intake from cruciferous vegetables, collected at recruitment via a semi-quantitative food frequency questionnaire, was combined with GSTM1, T1 and P1 genotype from peripheral blood lymphocytes or buccal mucosa. When categorized into high (greater than median) and low (less than/equal to median) intake, dietary ITC was slightly lower in cases than controls but the difference was not significant [odds ratio (OR) 0.81, 95% confidence interval (CI) 0.59-1.12]. There were no overall associations between GSTM1, T1 or P1 genotypes and colorectal cancer risk. However, among individuals with both GSTM1 and T1 null genotypes, we observed a 57% reduction in risk among high versus low consumers of ITC (OR 0.43, 95% CI 0.20-0.96), in particular for colon cancer (OR 0.31, 0.12-0.84). Our results are compatible with the hypothesis that ITCs from cruciferous vegetables modify risk of colorectal cancer in individuals with low GST activity. Further, this gene-diet interaction may be important in studies evaluating the effect of risk-enhancing compounds in the colorectum. PMID:12507929

  19. Glutathione S-transferase (GST) gene polymorphisms, cigarette smoking and colorectal cancer risk among Chinese in Singapore.

    PubMed

    Koh, Woon-Puay; Nelson, Heather H; Yuan, Jian-Min; Van den Berg, David; Jin, Aizhen; Wang, Renwei; Yu, Mimi C

    2011-10-01

    Cigarette smoking is a risk factor for colorectal cancer. Putative colorectal procarcinogens in tobacco smoke include polycyclic aromatic hydrocarbons and heterocyclic aromatic amines that are known substrates of glutathione S-transferases (GSTs). This study examined the influence of functional GST gene polymorphisms on the smoking-colorectal cancer association in a population known to be minimally exposed to dietary sources of these procarcinogens. Incident cases of colorectal cancer (n = 480) and matched controls (n = 1167) were selected from the Singapore Chinese Health Study, a population-based prospective cohort of 63 257 men and women who have been followed since 1993. We determined the deletion polymorphisms of GSTM1 and GSTT1 and the functional polymorphism at codon 105 of GSTP1 for each subject. A three level composite GST index was used to examine if GST profile affected a smoker's risk of developing colorectal cancer. While there was no statistically significant association between cigarette smoking and colorectal cancer risk among subjects absent of any at-risk GST genotypes, smokers possessing two to three at-risk GST genotypes exhibited a statistically significant increased risk of colorectal cancer compared with non-smokers (P = 0.0002). In this latter stratum, heavy smokers exhibited a >5-fold increased ris