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

  1. An endophytic taxol-producing fungus from Taxus x media, Aspergillus candidus MD3.

    PubMed

    Zhang, Peng; Zhou, Peng-Peng; Yu, Long-Jiang

    2009-04-01

    An endophytic taxol-producing fungus (strain MD3) isolated from the inner bark of Taxus x media was identified as Aspergillus candidus according to its morphological characteristics, physiological and biochemical characteristics, and 18S rRNA gene sequence analysis. Taxol produced by A. candidus MD3 was shown to be identical to authentic taxol analyzed by UV, HPLC, MS and nuclear magnetic resonance spectroscopy. The gene encoding the 10-deacetylbaccatin III-10-O-acetyl transferase, which catalyzes formation of the last diterpene intermediate in the taxol biosynthetic pathway, has been cloned from A. candidus MD3 for the first time and possesses high homology to the same gene found in Taxus spp.

  2. Fatal poisoning with Taxus baccata: quantification of paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol in body fluids by liquid chromatography-tandem mass spectrometry.

    PubMed

    Grobosch, T; Schwarze, B; Stoecklein, D; Binscheck, T

    2012-01-01

    This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid-liquid extraction under alkaline conditions followed by LC-MS-MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 × 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohydroxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantitative evaluation revealed taxoid concentrations ranging from 4.5 to 132 µg/L (stomach content), 1 to 200 µg/L (urine), <0.5 to 12 µg/L (cardiac blood), <0.5 to 7.3 µg/L (femoral blood), and 4.9 to 290 µg/L (bile). In brain tissue, none of these taxoids could be detected (<0.5 µg/L). In urine, after enzymatic hydrolysis, the concentration of 3,5-dimethoxyphenol (3,5-DMP) was 23,000 µg/L. The alkaloidal diterpenoids were found in all postmortem samples. The newly developed LC-MS-MS method enables the identification of alkaloidal and non-alkaloidal diterpenoids and 3,5-dimethoxyphenol in human body fluids and tissues for the confirmation of accidental or intentional poisonings with yew plant material.

  3. An endophytic taxol-producing fungus from Taxus media, Cladosporium cladosporioides MD2.

    PubMed

    Zhang, Peng; Zhou, Peng-Peng; Yu, Long-Jiang

    2009-09-01

    Fermentation processes using taxol-producing fungi other than Taxus spp. may be an alternative way to produce taxol, which is an important antitumor agent used widely in the clinic setting. In this study, a taxol-producing endophytic fungus strain MD2 was isolated from the inner bark of Taxus media. Strain MD2 produced taxol when grown in potato dextrose liquid medium. The fungal taxol-which was analyzed by ultraviolet, high-performance liquid chromatography and mass spectrometry-was shown to be identical to authentic taxol and 10-deacetylbaccatin III. Further analysis with nuclear magnetic resonance (NMR) spectroscopy to show the chemical structure of the fungal taxol indicated that the fungal taxol produced an NMR spectrum identical to that of authentic taxol. Strain MD2 was identified as Cladosporium cladosporioides according to morphology of the fungal culture, characteristics of the spores, and analysis of 18S rDNA sequence. In addition, 10-deacetylbaccatin III-10-O-acetyl transferase gene of C. cladosporioides MD2 was cloned for the first time and was shown to share 99% identity with that of T. x media and 97% identity with that of T. wallichiana var. mairei.

  4. Glutathione transferases and neurodegenerative diseases.

    PubMed

    Mazzetti, Anna Paola; Fiorile, Maria Carmela; Primavera, Alessandra; Lo Bello, Mario

    2015-03-01

    There is substantial agreement that the unbalance between oxidant and antioxidant species may affect the onset and/or the course of a number of common diseases including Parkinson's and Alzheimer's diseases. Many studies suggest a crucial role for oxidative stress in the first phase of aging, or in the pathogenesis of various diseases including neurological ones. Particularly, the role exerted by glutathione and glutathione-related enzymes (Glutathione Transferases) in the nervous system appears more relevant, this latter tissue being much more vulnerable to toxins and oxidative stress than other tissues such as liver, kidney or muscle. The present review addresses the question by focusing on the results obtained by specimens from patients or by in vitro studies using cells or animal models related to Parkinson's and Alzheimer's diseases. In general, there is an association between glutathione depletion and Parkinson's or Alzheimer's disease. In addition, a significant decrease of glutathione transferase activity in selected areas of brain and in ventricular cerebrospinal fluid was found. For some glutathione transferase genes there is also a correlation between polymorphisms and onset/outcome of neurodegenerative diseases. Thus, there is a general agreement about the protective effect exerted by glutathione and glutathione transferases but no clear answer about the mechanisms underlying this crucial role in the insurgence of neurodegenerative diseases.

  5. Feruloyl-CoA:monolignol transferase

    DOEpatents

    Wilkerson, Curtis; Ralph, John; Withers, Saunia; Mansfield, Shawn D.

    2016-09-13

    The invention relates to nucleic acids encoding a feruloyl-CoA:monolignol transferase and the feruloyl-CoA:monolignol transferase enzyme that enables incorporation of monolignol ferulates, for example, including p-coumaryl ferulate, coniferyl ferulate, and sinapyl ferulate, into the lignin of plants.

  6. Glutathione transferases: a structural perspective.

    PubMed

    Oakley, Aaron

    2011-05-01

    The glutathione transferases (GSTs) are one of the most important families of detoxifying enzymes in nature. The classic activity of the GSTs is conjugation of compounds with electrophilic centers to the tripeptide glutathione (GSH), but many other activities are now associated with GSTs, including steroid and leukotriene biosynthesis, peroxide degradation, double-bond cis-trans isomerization, dehydroascorbate reduction, Michael addition, and noncatalytic "ligandin" activity (ligand binding and transport). Since the first GST structure was determined in 1991, there has been an explosion in structural data across GSTs of all three families: the cytosolic GSTs, the mitochondrial GSTs, and the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG family). In this review, the major insights into GST structure and function will be discussed.

  7. [Structure and functions of glutathione transferases].

    PubMed

    Fedets, O M

    2014-01-01

    Data about classification, nomenclature, structure, substrate specificity and role of many glutathione transferase's isoenzymes in cell functions have been summarised. The enzyme has been discovered more than 50 years ago. This family of proteins is updated continuously. It has very different composition and will have demand for system analysis for many years.

  8. Differentiation Between Intracellular and Cell Surface Glycosyl Transferases: Galactosyl Transferase Activity in Intact Cells and in Cell Homogenate

    PubMed Central

    Deppert, Wolfgang; Werchau, Hermann; Walter, Gernot

    1974-01-01

    Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition. PMID:4528509

  9. 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

  10. 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.

  11. Glutathione transferase gene family from the housefly Musca domestica.

    PubMed

    Syvanen, M; Zhou, Z H; Wang, J Y

    1994-10-17

    Three new glutathione transferase (GST) genes from the housefly Musca domestica are described. These genes, identified as MdGST-2, -3, and -4, were from cDNA clones obtained from a cDNA bank in phage lambda. The bank was prepared using poly(A)+ RNA from a housefly that is highly resistant to organophosphate insecticides because of enhanced expression of multiple members of the glutathione transferase gene family. The DNA sequence of each is reported and has a complete open reading frame that specified an amino acid sequence similar to other dipteran glutathione transferases. Based on phylogenetic analysis, we can conclude that the insect glutathione transferase gene family falls into two groups, each of which evolves at a different rate, presumably due to differences in functional constraints. We show that MdGST-1 (and their homologues from Drosophila and Lucilia) evolve at a significantly slower rate than the other members of the gene family. Each housefly GST cDNA was inserted into a bacterial plasmid expression system and a glutathione transferase activity was expressed in Escherichia coli. The transcription pattern of each of these glutathione transferases was examined in a variety of different housefly strains that are known to differ in their resistance to organophosphate insecticides due to different patterns of glutathione transferase expression. We found that the level of transcription for two of our clones was positively correlated with the level of organophosphate resistance.

  12. 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

  13. Hypoxanthine-guanine phosphoribosyl transferase deficiency.

    PubMed

    de Bruyn, C H

    1976-02-29

    In man congential lack of enzyme of the purine salvage system, hypoxanthineguanine phosphoribosyl transferase (HG-PRT E.C. 2.4.2.8), is mostly accompanied by a picture known as the Lesch-Nyhan snydrome. The degree of deficiency may vary from zero to a few percent of normal activity but a correlation between the severity of HG-PRT deficiency and the clinical picture has not been observed, no more than a correlation HG-PRT deficiency and neurological dysfunction. But individuals with undetectable HG-PRT activity but without the Lesch-Nyhan syndrome have been described. Patients with partial HG-PRT defiency have clinically distinctive findings. Sometimes mild neurological abnormalities are observed. Because of marked overproduction of ric acid severe gouty arthritis and renal dysfunction are often encountered in both complete and partial deficiency. There is considerable molecular heterogeneity in HG-PRT deficiency in man. Mutant ebnzymes may exhibit different kinetic and electrophoretic properties, indicating that hterwe might be a mutation on the structural gene coding for HG-PRT. Lack of HG-PRT disturbs purine interconversions profoundly. In addition to an important function of HG-PRT in the uptake of the purine hypoxantine and guanine into the cell, the effective uptake of inosine, guanosine and adenosine also seems to be dependent on HG-PRT...

  14. Glutathione transferases in the bioactivation of azathioprine.

    PubMed

    Modén, Olof; Mannervik, Bengt

    2014-01-01

    The prodrug azathioprine is primarily used for maintaining remission in inflammatory bowel disease, but approximately 30% of the patients suffer adverse side effects. The prodrug is activated by glutathione conjugation and release of 6-mercaptopurine, a reaction most efficiently catalyzed by glutathione transferase (GST) A2-2. Among five genotypes of GST A2-2, the variant A2*E has threefold-fourfold higher catalytic efficiency with azathioprine, suggesting that the expression of A2*E could boost 6-mercaptopurine release and adverse side effects in treated patients. Structure-activity studies of the GST A2-2 variants and homologous alpha class GSTs were made to delineate the determinants of high catalytic efficiency compared to other alpha class GSTs. Engineered chimeras identified GST peptide segments of importance, and replacing the corresponding regions in low-activity GSTs by these short segments produced chimeras with higher azathioprine activity. By contrast, H-site mutagenesis led to decreased azathioprine activity when active-site positions 208 and 213 in these favored segments were mutagenized. Alternative substitutions indicated that hydrophobic residues were favored. A pertinent question is whether variant A2*E represents the highest azathioprine activity achievable within the GST structural framework. This issue was addressed by mutagenesis of H-site residues assumed to interact with the substrate based on molecular modeling. The mutants with notably enhanced activities had small or polar residues in the mutated positions. The most active mutant L107G/L108D/F222H displayed a 70-fold enhanced catalytic efficiency with azathioprine. The determination of its structure by X-ray crystallography showed an expanded H-site, suggesting improved accommodation of the transition state for catalysis.

  15. 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

  16. 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.

  17. Homogentisate solanesyl transferase (HST) cDNA’s in maize

    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 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 reaction ...

  18. 21 CFR 862.1535 - Ornithine carbamyl transferase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

  19. Histamine N-methyl transferase: inhibition by drugs.

    PubMed Central

    Pacifici, G M; Donatelli, P; Giuliani, L

    1992-01-01

    1. Histamine N-methyl transferase activity was measured in samples of human liver, brain, kidney, lung and intestinal mucosa. The mean (+/- s.d.) rate (nmol min-1 mg-1 protein) of histamine N-methylation was 1.78 +/- 0.59 (liver, n = 60), 1.15 +/- 0.38 (renal cortex, n = 8), 0.79 +/- 0.14 (renal medulla, n = 8), 0.35 +/- 0.08 (lung, n = 20), 0.47 +/- 0.18 (human intestine, n = 30) and 0.29 +/- 0.14 (brain, n = 13). 2. Inhibition of histamine N-methyl transferase by 15 drugs was investigated in human liver. The IC50 for the various drugs ranged over three orders of magnitude; chloroquine was the most potent inhibitor. 3. The average IC50 values for chloroquine were 12.6, 22.0, 19.0, 21.6 microM in liver, renal cortex, brain and colon, respectively. These values are lower than the Michaelis-Menten constant for histamine N-methyltransferase in liver (43.8 microM) and kidney (45.5 microM). Chloroquine carried a mixed non-competitive inhibition of hepatic histamine N-methyl transferase. Some side-effects of chloroquine may be explained by inhibition of histamine N-methyl transferase. PMID:1457266

  20. 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 ...

  1. Phosphorylation and inhibition of. gamma. -glutamyl transferase activity by cAMP-dependent protein kinase

    SciTech Connect

    Kolesnichenko, L.S.; Chernov, N.N.

    1986-10-20

    It was shown that preparations of bovine kidney ..gamma..-glutamyl transferase of differing degrees of purity are phosphorylated by cAMP-dependent protein kinase. This is accompanied by a decrease in both the transferase and hydrolase activities of the enzyme. Consequently, ..gamma..-glutamyl transferase may serve as the substrate and target of the regulation of cAMP-dependent protein kinase.

  2. Proton mobilities in crambin and glutathione S-transferase

    NASA Astrophysics Data System (ADS)

    Wanderlingh, U. N.; Corsaro, C.; Hayward, R. L.; Bée, M.; Middendorf, H. D.

    2003-08-01

    Using a neutron backscattering spectrometer, the temperature dependence of mean-square atomic displacements derived from window-integrated quasielastic spectra was measured for two D 2O-hydrated proteins: crambin and glutathione S-transferase. Analyses show that the anharmonic dynamics observed around and above 200 K is consistent with a description in terms of proton/deuteron jumps within asymmetric double-minimum potentials. Also determined were activation energies along with estimates of effective masses and average oscillator energies.

  3. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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)...

  4. 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)...

  5. 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)...

  6. Electrochemical evaluation of glutathione S-transferase kinetic parameters.

    PubMed

    Enache, Teodor Adrian; Oliveira-Brett, Ana Maria

    2015-02-01

    Glutathione S-transferases (GSTs), are a family of enzymes belonging to the phase II metabolism that catalyse the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds. The voltammetric behaviour of glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione S-transferase (GST), as well as the catalytic conjugation reaction of GSH to CDNB by GST was investigated at room temperature, T=298.15K (25°C), at pH6.5, for low concentration of substrates and enzyme, using differential pulse (DP) voltammetry at a glassy carbon electrode. Only GSH can be oxidized; a sensitivity of 0.14nA/μM and a LOD of 6.4μM were obtained. The GST kinetic parameter electrochemical evaluation, in relation to its substrates, GSH and CDNB, using reciprocal Michaelis-Menten and Lineweaver-Burk double reciprocal plots, was determined. A value of KM~100μM was obtained for either GSH or CDNB, and Vmax varied between 40 and 60μmol/min per mg of GST.

  7. Elevation of alanine amino transferase and aspartate amino transferase produced by pyoverdin, a photolabile pigment of Pseudomonas fluorescens.

    PubMed

    Eraso, A J; Albesa, I

    1998-01-01

    The effect of three forms pyoverdin on mouse liver was studied. Significant increases of alanine amino transferase (ALT) and aspartate amino transferase (AST) were obtained in mice after ingestion of water with forms A and C. The effect on liver was more evident with A than with C. Pyoverdin was purified by means of salt saturation, solvent extractions and ion-exchange chromatography. Fluorescent peaks obtained in the presence of light were different from those eluted under dark conditions. The relative amounts of pyoverdin A, B and C varied when dark purification procedure was employed. Form A decreased while C increased in the absence of light. Optimum conditions for C were in the dark without iron. When C was exposed to light, it changed to form A. Fast Atom Bombardment (FAB) mass spectrometry of pyoverdin form C gave a form at M+ = 1324 m.u., which is 9 m.u. less than pyoverdin purified in the presence of light. The results suggest that light can influence pyoverdin stability and toxicity. PMID:9888631

  8. Geranylgeranyl transferase type II inhibition prevents myeloma bone disease.

    PubMed

    Lawson, Michelle A; Coulton, Les; Ebetino, Frank H; Vanderkerken, Karin; Croucher, Peter I

    2008-12-12

    Geranylgeranyl transferase II (GGTase II) is an enzyme that plays a key role in the isoprenylation of proteins. 3-PEHPC, a novel GGTase II inhibitor, blocks bone resorption and induces myeloma cell apoptosis in vitro. Its effect on bone resorption and tumor growth in vivo is unknown. We investigated the effect of 3-PEHPC on tumor burden and bone disease in the 5T2MM model of multiple myeloma in vivo. 3-PEHPC significantly reduced osteoclast numbers and osteoclast surface. 3-PEHPC prevented the bone loss and the development of osteolytic bone lesions induced by 5T2MM myeloma cells. Treatment with 3-PEHPC also significantly reduced myeloma burden in bone. The magnitude of response was similar to that seen with the bisphosphonate, risedronate. These data show that targeting GGTase II with 3-PEHPC can prevent osteolytic bone disease and reduce tumor burden in vivo, and represents a novel approach to treating tumors that grow in bone.

  9. Pleiotropic Functions of Glutathione S-Transferase P

    PubMed Central

    Zhang, Jie; Grek, Christina; Ye, Zhi-Wei; Manevich, Yefim; Tew, Kenneth D.; Townsend, Danyelle M.

    2016-01-01

    Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation. The expression of GSTP has been linked with cancer and other human pathologies and more recently even with drug addiction. With respect to human health, polymorphic variants of GSTP may determine individual susceptibility to oxidative stress and/or be critical in the design and development of drugs that have used redox pathways as a discovery platform. PMID:24974181

  10. Glutathione analogue sorbents selectively bind glutathione S-transferase isoenzymes.

    PubMed

    Castro, V M; Kelley, M K; Engqvist-Goldstein, A; Kauvar, L M

    1993-06-01

    Novel affinity sorbents for glutathione S-transferases (GSTs) were created by binding glutathione (GSH) analogues to Sepharose 6B. The GSH molecule was modified at the glycine moiety and at the group attached to the sulphur of cysteine. When tested by affinity chromatography in a flow-through microplate format, several of these sorbents selectively bound GST isoenzymes. gamma E-C(Hx)-phi G (glutathione with a hexyl moiety bound to cysteine and phenylglycine substituted for glycine) specifically bound rat GST 7-7, the Pi-class isoenzyme, from liver, kidney and small intestine. gamma E-C(Bz)-beta A (benzyl bound to cysteine and beta-alanine substituted for glycine) was highly selective for rat subunits 3 and 4, which are Mu-class isoenzymes. By allowing purification of the isoenzymes under mild conditions that preserve activity, the novel sorbents should be useful in characterizing the biological roles of GSTs in both normal animal and cancer tissues.

  11. 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.

  12. Pleiotropic functions of glutathione S-transferase P.

    PubMed

    Zhang, Jie; Grek, Christina; Ye, Zhi-Wei; Manevich, Yefim; Tew, Kenneth D; Townsend, Danyelle M

    2014-01-01

    Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation. The expression of GSTP has been linked with cancer and other human pathologies and more recently even with drug addiction. With respect to human health, polymorphic variants of GSTP may determine individual susceptibility to oxidative stress and/or be critical in the design and development of drugs that have used redox pathways as a discovery platform.

  13. Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

    PubMed Central

    Parales, R E; Harwood, C S

    1992-01-01

    beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified. Images PMID:1624453

  14. 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

  15. 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

  16. 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.

  17. Mannosyl transferase activity in homogenates of adult Schistosoma mansoni.

    PubMed

    Rumjanek, F D; Smithers, S R

    1978-08-01

    Homogenates of adult Schistosoma mansoni contain enzymes which are capable of transferring [14C]mannose from GDP[U-14C]mannose to a lipid acceptor which migrates as a single peak on a silica gel thin-layer plate. This lipid may belong to the class of polyprenol monophosphates which are intermediate elements in the glycosylation of nascent proteins. The schistosome mannosyl transferase activity is associated with membranous particles and is dependent on the presence of Mn2+. However, other divalent metals such as Mg2+ or Ca2+ can, in decreasing order of efficiency, replace Mn2+. When UDP[U-14C]glucose was incubated with the homogenates in the same conditions, relatively little label was transferred to the lipid acceptor. Live worms incubated in a medium containing GDP[U-14C]mannose seem to incorporate the label preferentially on the tegument and on adjacent subtegumental structures. By adding foetal calf serum to the medium, incorporation of the label can be stimulated.

  18. Glutathione S-transferase, incense burning and asthma in children.

    PubMed

    Wang, I-J; Tsai, C-H; Chen, C-H; Tung, K-Y; Lee, Y L

    2011-06-01

    Incense burning is a popular practice in many family homes and temples. However, little is known about the effects of indoor incense burning and genetic polymorphisms on asthma. This study evaluated the effects of indoor incense burning and glutathione S-transferase (GST) genetic polymorphisms on asthma and wheeze. In 2007, 3,764 seventh-grade schoolchildren (mean±sd age 12.42±0.65 yrs) were evaluated using a standard questionnaire for information about respiratory symptoms and environmental exposures. Multiple logistic regressions were performed to assess the association between GST polymorphisms and incense burning frequency on asthma and wheeze, after adjusting for potential confounders. The frequency of incense burning at home was associated with increased risk of current asthma (p=0.05), medication use (p=0.03) and exercise wheeze (p=0.001). GST1 (GSTT1) null genotypes were associated with current asthma (OR 1.43, 95% CI 1.00-2.04) and medication use (OR 1.46, 95% CI 1.01-2.22). GSTT1 showed a significant interactive effect with incense burning on current asthma, current wheeze and nocturnal wheeze. The frequency of incense burning was associated with increased risk of current asthma, medication use, lifetime wheeze, nocturnal wheeze and exercise wheeze in an exposure-response manner among children with GSTT1 null genotype (p<0.05). Incense burning is a risk factor for asthma and wheezing, especially in GSTT1 genetically susceptible children.

  19. Glucuronyl transferase deficiency and mild hereditary spherocytosis: effect of splenectomy.

    PubMed

    Eber, S W; Ullrich, D; Speer, C P; Armbrust, R; Schröter, W

    1988-08-01

    In a 6-year-old girl an association of hereditary spherocytosis and a defect in hepatic bilirubin metabolism has been found. The patient suffered from mild compensated haemolytic anaemia and excessive hyperbilirubinaemia (maximum concentration 581 mumol/l), the serum activity of liver enzymes was slightly increased. Examination of the erythrocyte membrane proteins revealed a deficiency of the major membrane skeletal protein, spectrin (about 75% of normal) which is probably the basic genetic defect of hereditary spherocytosis. Examination of the patient's family revealed a recessive mode of inheritance. The concentration of bilirubin conjugates in the patient's serum was decreased due to a reduced UDP-glucuronyl transferase activity found in homogenates of liver tissue. Histological liver examination showed an intrahepatic cholestasis, which is a secondary and reversible alteration resulting from severe hyperbilirubinaemia. After splenectomy, normalization of the increased haemolysis and hepatic dysfunction was observed. The excessive hyperbilirubinaemia can be explained by the association of an increased bilirubin load due to haemolytic anaemia and the diminished hepatic conjugation of bilirubin.

  20. The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase.

    PubMed

    Fung, Angela Wai Shan; Leung, Charles Chung Yun; Fahlman, Richard Peter

    2014-08-01

    Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3' aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA(Leu) (anticodon 5'-CAG-3') isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA(Leu) (CAG)-a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)-that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.

  1. The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

    PubMed Central

    Fung, Angela Wai Shan; Leung, Charles Chung Yun; Fahlman, Richard Peter

    2014-01-01

    Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNALeu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNALeu (CAG)—a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed. PMID:24935875

  2. 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

  3. Glutathione transferase classes alpha, pi, and mu: GSH activation mechanism.

    PubMed

    Dourado, Daniel F A R; Fernandes, Pedro Alexandrino; Ramos, Maria João

    2010-10-14

    Since the early 1960s, glutathione transferases (GSTs) have been described as detoxification enzymes. In fact, GSTs are the most important enzymes involved in the metabolism of electrophilic xenobiotic/endobiotic compounds. These enzymes are able to catalyze the nucleophilic addition of glutathione (GSH) sulfur thiolate to a wide range of electrophilic substrates, building up a less toxic and more soluble compound. Cytosolic classes alpha, pi, and mu are the most extensively studied GSTs. However, many of the catalytic events are still poorly understood. In the present work, we have resorted to density functional theory (DFT) and to potential of mean force (PMF) calculations to determine the GSH activation mechanism of GSTP1-1 and GSTM1-1 isoenzymes. For the GSTP1-1 enzyme, we have demonstrated that a water molecule, after an initial conformational rearrangement of GSH, can assist a proton transfer between the GSH cysteine thiol (GSH-SH) and the GSH glutamate alpha carboxylate (GSH-COO(-)) groups. The energy barrier associated with the proton transfer is 11.36 kcal·mol(-1). The GSTM1-1 enzyme shows a completely different behavior from the previous isoenzyme. In this case, two water molecules, positioned between the GSH-SH and the ξ N atom of His107, working like a bridge, are able to promote the proton transfer between these two active groups with an energy barrier of 7.98 kcal·mol(-1). All our results are consistent with all the enzymes kinetics and mutagenesis experimental studies.

  4. Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization.

    PubMed

    van Grinsven, Koen W A; Rosnowsky, Silke; van Weelden, Susanne W H; Pütz, Simone; van der Giezen, Mark; Martin, William; van Hellemond, Jaap J; Tielens, Aloysius G M; Henze, Katrin

    2008-01-18

    Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles. PMID:18024431

  5. Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

    PubMed

    Glisic, Branka; Mihaljevic, Ivan; Popovic, Marta; Zaja, Roko; Loncar, Jovica; Fent, Karl; Kovacevic, Radmila; Smital, Tvrtko

    2015-01-01

    Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 μM (Gstt1a) to 250 μM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the

  6. Unusual metal ion catalysis in an acyl-transferase ribozyme.

    PubMed

    Suga, H; Cowan, J A; Szostak, J W

    1998-07-14

    Most studies of the roles of catalytic metal ions in ribozymes have focused on inner-sphere coordination of the divalent metal ions to the substrate or ribozyme. However, divalent metal ions are strongly hydrated in water, and some proteinenzymes, such as Escherichia coli RNase H and exonuclease III, are known to use metal cofactors in their fully hydrated form [Duffy, T. H., and Nowak, T. (1985) Biochemistry 24, 1152-1160; Jou, R., and Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685-6686]. It is therefore important to consider the possibility of outer-sphere coordination of catalytic metal ions in ribozymes. We have used an exchange-inert metal complex, cobalt hexaammine, to show that the catalytic metal ion in an acyl-transferase ribozyme acts through outer-sphere coordination. Our studies provide an example of a fully hydrated Mg2+ ion that plays an essential role in ribozyme catalysis. Kinetic studies of wild-type and mutant ribozymes suggest that a pair of tandem G:U wobble base pairs adjacent to the reactive center constitute the metal-binding site. This result is consistent with recent crystallographic studies [Cate, J. H., and Doudna, J. A. (1996) Structure 4, 1221-1229; Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685; Cate, J. H., Hanna, R. L., and Doudna, J. A. (1997) Nat. Struct. Biol. 4, 553-558] showing that tandem wobble base pairs are good binding sites for metal hexaammines. We propose a model in which the catalytic metal ion is bound in the major groove of the tandem wobble base pairs, is precisely positioned by the ribozyme within the active site, and stabilizes the developing oxyanion in the transition state. Our results may have significant implications for understanding the mechanism of protein synthesis [Noller, H. F., Hoffarth, V., and Zimniak, L. (1992) Science 256, 1416-1419].

  7. Peptidyl transferase inhibition by the nascent leader peptide of an inducible cat gene.

    PubMed Central

    Gu, Z; Rogers, E J; Lovett, P S

    1993-01-01

    The site of ribosome stalling in the leader of cat transcripts is critical to induction of downstream translation. Site-specific stalling requires translation of the first five leader codons and the presence of chloramphenicol, a sequence-independent inhibitor of ribosome elongation. We demonstrate in this report that a synthetic peptide (the 5-mer) corresponding to the N-terminal five codons of the cat-86 leader inhibits peptidyl transferase in vitro. The N-terminal 2-, 3-, and 4-mers and the reverse 5-mer (reverse amino acid sequence of the 5-mer) are virtually without effect on peptidyl transferase. A missense mutation in the cat-86 leader that abolishes induction in vivo corresponds to an amino acid replacement in the 5-mer that completely relieves peptidyl transferase inhibition. In contrast, a missense mutation that does not interfere with in vivo induction corresponds to an amino acid replacement in the 5-mer that does not significantly alter peptidyl transferase inhibition. Our results suggest that peptidyl transferase inhibition by the nascent cat-86 5-mer peptide may be the primary determinant of the site of ribosome stalling in the leader. A model based on this concept can explain the site specificity of ribosome stalling as well as the response of induction to very low levels of the antibiotic inducer. Images PMID:7690023

  8. Structure of succinyl-CoA:3-ketoacid CoA transferase from Drosophila melanogaster

    PubMed Central

    Zhang, Min; Xu, Han-Yang; Wang, Yi-Cui; Shi, Zhu-Bing; Zhang, Nan-Nan

    2013-01-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT) plays a crucial role in ketone-body metabolism. SCOT from Drosophila melanogaster (DmSCOT) was purified and crystallized. The crystal structure of DmSCOT was determined at 2.64 Å resolution and belonged to space group P212121, with unit-cell parameters a = 76.638, b = 101.921, c = 122.457 Å, α = β = γ = 90°. Sequence alignment and structural analysis identified DmSCOT as a class I CoA transferase. Compared with Acetobacter aceti succinyl-CoA:acetate CoA transferase, DmSCOT has a different substrate-binding pocket, which may explain the difference in their substrate specificities. PMID:24100554

  9. Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo.

    PubMed

    Song, Lina; Bachert, Collin; Linstedt, Adam D

    2016-01-01

    Mucin-type O-glycosylation occurring in the Golgi apparatus is an important protein posttranslational modification initiated by up to 20 GalNAc-transferase isozymes with largely distinct substrate specificities. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and misregulation causes human diseases. Here we describe the use of protein-based fluorescence sensors that traffic in the secretory pathway to monitor GalNAc-transferase activity in living cells. The sensors can either be "pan" or isozyme specific.

  10. Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo.

    PubMed

    Song, Lina; Bachert, Collin; Linstedt, Adam D

    2016-01-01

    Mucin-type O-glycosylation occurring in the Golgi apparatus is an important protein posttranslational modification initiated by up to 20 GalNAc-transferase isozymes with largely distinct substrate specificities. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and misregulation causes human diseases. Here we describe the use of protein-based fluorescence sensors that traffic in the secretory pathway to monitor GalNAc-transferase activity in living cells. The sensors can either be "pan" or isozyme specific. PMID:27632006

  11. 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…

  12. 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°.

  13. 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

  14. 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...

  15. Glutathione S-transferase class mu in French alcoholic cirrhotic patients.

    PubMed

    Groppi, A; Coutelle, C; Fleury, B; Iron, A; Begueret, J; Couzigou, P

    1991-09-01

    The lack of glutathione S-transferase mu (GST mu) was examined in 45 healthy French Caucasians and 45 alcoholic cirrhotic French Caucasians: microsamples of blood were taken and DNA amplified by the polymerase chain reaction. We have concluded that there is no relationship between this genotype and the development of alcoholic cirrhosis in these heavy consumers of ethanol.

  16. 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...

  17. 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...

  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. Development of isoform-specific sensors of polypeptide GalNAc-transferase activity.

    PubMed

    Song, Lina; Bachert, Collin; Schjoldager, Katrine T; Clausen, Henrik; Linstedt, Adam D

    2014-10-31

    Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Here we describe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitro work. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.

  2. 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....

  3. 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...

  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 Clinical Chemistry Test Systems § 862.1315...

  5. A practical fluorogenic substrate for high-throughput screening of glutathione S-transferase inhibitors.

    PubMed

    Fujikawa, Yuuta; Morisaki, Fumika; Ogura, Asami; Morohashi, Kana; Enya, Sora; Niwa, Ryusuke; Goto, Shinji; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Inoue, Hideshi

    2015-07-21

    We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.

  6. 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.

  7. 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...

  8. 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...

  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 862.1100 - 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. 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

  19. Purification and Characterization of the Staphylococcus aureus Bacillithiol Transferase BstA

    PubMed Central

    Perera, Varahenage R.; Newton, Gerald L.; Parnell, Jonathan M.; Komives, Elizabeth A.; Pogliano, Kit

    2016-01-01

    Background Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins. Methods We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays. Results BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity. Conclusions BstA is a bacillithiol transferase from Staphylococcus aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules. General Significance BstA is an active bacillithiol transferase from Staphylococcus aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from Bacillus subtilis YfiT. PMID:24821014

  20. 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.

  1. 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.

  2. 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

  3. Effects of 2(3)-tert-butyl-4-hydroxyanisole pretreatment on cefpiramide binding to mouse glutathione S-transferases.

    PubMed

    Nishiya, H; Haga, T; Nozue, N; Komatsu, T; Baba, M; Ueda, Y; Ono, Y; Kunii, O

    1989-01-01

    Binding of cefpiramide (CPM) and other beta-lactam antimicrobial agents to 2(3)-tert-butyl-4-hydroxyanisole (BHA)-induced liver glutathione (GSH) S-transferases (EC 2.5.1.18) from CD-1 mice was studied. A marked induction of hepatic GSH S-transferase from mice fed BHA was observed. Gel chromatography of liver cytosol from mice fed BHA showed an increased binding of CPM, cefotetan and cefazolin to BHA-induced GSH S-transferases. The extent of their binding to GSH S-transferase seemed to be correlated with the extent of their excretion into the bile. Binding of CPM to the GSH S-transferase fraction was inhibited by both indocyanine green, which is known to bind liver GSH S-transferases intensively, and by cefoperazon, which is mainly excreted into the bile. This study suggests that GSH S-transferases are the main binding proteins of CPM in the liver cytosol fraction and play an important role as carrier proteins of CPM and some antimicrobial agents in mouse liver.

  4. GalNAc-transferase specificity prediction based on feature selection method.

    PubMed

    Lu, Lin; Niu, Bing; Zhao, Jun; Liu, Liang; Lu, Wen-Cong; Liu, Xiao-Jun; Li, Yi-Xue; Cai, Yu-Dong

    2009-02-01

    GalNAc-transferase can catalyze the biosynthesis of O-linked oligosaccharides. The specificity of GalNAc-transferase is composed of nine amino acid residues denoted by R4, R3, R2, R1, R0, R1', R2', R3', R4'. To predict whether the reducing monosaccharide will be covalently linked to the central residue R0(Ser or Thr), a new method based on feature selection has been proposed in our work. 277 nonapeptides from reference [Chou KC. A sequence-coupled vector-projection model for predicting the specificity of GalNAc-transferase. Protein Sci 1995;4:1365-83] are chosen for training set. Each nonapeptide is represented by hundreds of amino acid properties collected by Amino Acid Index database (http://www.genome.jp/aaindex) and transformed into a numeric vector with 4554 features. The Maximum Relevance Minimum Redundancy (mRMR) method combining with Incremental Feature Selection (IFS) and Feature Forward Selection (FFS) are then applied for feature selection. Nearest Neighbor Algorithm (NNA) is used to build prediction models. The optimal model contains 54 features and its correct rate tested by Jackknife cross-validation test reaches 91.34%. Final feature analysis indicates that amino acid residues at position R3' play the most important role in the recognition of GalNAc-transferase specificity, which were confirmed by the experiments [Elhammer AP, Poorman RA, Brown E, Maggiora LL, Hoogerheide JG, Kezdy FJ. The specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase as inferred from a database of in vivo substrates and from the in vitro glycosylation of proteins and peptides. J Biol Chem 1993;268:10029-38; O'Connell BC, Hagen FK, Tabak LA. The influence of flanking sequence on the O-glycosylation of threonine in vitro. J Biol Chem 1992;267:25010-8; Yoshida A, Suzuki M, Ikenaga H, Takeuchi M. Discovery of the shortest sequence motif for high level mucin-type O-glycosylation. J Biol Chem 1997;272:16884-8]. Our method can be used as a tool for predicting O

  5. Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1).

    PubMed

    Orchard, Michael G; Neuss, Judi C; Galley, Carl M S; Carr, Andrew; Porter, David W; Smith, Phillip; Scopes, David I C; Haydon, David; Vousden, Katherine; Stubberfield, Colin R; Young, Kate; Page, Martin

    2004-08-01

    The first inhibitors of fungal protein: mannosyl transferase 1 (PMT1) are described. They are based upon rhodanine-3-acetic acid and several compounds have been identified, for example, 5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic acid (5a), which inhibit Candida albicans PMT1 with IC(50)s in the range 0.2-0.5 microM. Members of the series are effective in inducing changes in morphology of C. albicans in vitro that have previously been associated with loss of the transferase activity. These compounds could serve as useful tools for studying the effects of protein O-mannosylation and its relevance in the search for novel antifungal agents. PMID:15225710

  6. Design, synthesis, and characterization of peptide-based rab geranylgeranyl transferase inhibitors.

    PubMed

    Tan, Kui-Thong; Guiu-Rozas, Ester; Bon, Robin S; Guo, Zhong; Delon, Christine; Wetzel, Stefan; Arndt, Sabine; Alexandrov, Kirill; Waldmann, Herbert; Goody, Roger S; Wu, Yao-Wen; Blankenfeldt, Wulf

    2009-12-24

    Rab geranylgeranyl transferase (RabGGTase) catalyzes the attachment of geranylgeranyl isoprenoids to Rab guanine triphosphatases, which are key regulators in vesicular transport. Because geranylgeranylation is required for proper function and overexpression of Rabs has been observed in various cancers, RabGGTase may be a target for novel therapeutics. The development of selective inhibitors is, however, difficult because two related enzymes involved in other cellular processes exist in eukaryotes and because RabGGTase recognizes protein substrates indirectly, resulting in relaxed specificity. We report the synthesis of a peptidic library based on the farnesyl transferase inhibitor pepticinnamin E. Of 469 compounds investigated, several were identified as selective for RabGGTase with low micromolar IC(50) values. The compounds were not generally cytotoxic and inhibited Rab isoprenylation in COS-7 cells. Crystal structure analysis revealed that selective inhibitors interact with a tunnel unique to RabGGTase, implying that this structural motif is an attractive target for improved RabGGTase inhibitors.

  7. New members of the glutathione transferase family discovered in red and brown algae.

    PubMed

    Hervé, Cécile; de Franco, Pierre-Olivier; Groisillier, Agnès; Tonon, Thierry; Boyen, Catherine

    2008-06-15

    The GSTs (glutathione transferases) are involved in the detoxification of a wide variety of hydrophobic substrates. These enzymes have been found in virtually all types of organisms, including plants, animals, nematodes and bacteria. In the present study, we report the molecular and biochemical characterization of algal GSTs. Phylogenetic analysis showed that most of them were distinct from previously described GST classes, but were most closely related to the Sigma class. Profiling of GST genes from the red alga Chondrus crispus and brown alga Laminaria digitata was undertaken after different chemical treatments and showed that they displayed contrasting patterns of transcription. Recombinant algal GST from both species showed transferase activities against the common substrates aryl halides, but also on the alpha,beta-unsaturated carbonyl 4-hydroxynonenal. Also, they exhibit significant peroxidation towards organic hydroperoxides, including oxygenated derivatives of polyunsaturated fatty acids. Among a range of compounds tested, Cibacron Blue was the most efficient inhibitor of algal GSTs identified.

  8. Three-dimensional structure of a Bombyx mori Omega-class glutathione transferase.

    PubMed

    Yamamoto, Kohji; Suzuki, Mamoru; Higashiura, Akifumi; Nakagawa, Atsushi

    2013-09-01

    Glutathione transferases (GSTs) are major phase II detoxification enzymes that play central roles in the defense against various environmental toxicants as well as oxidative stress. Here we report the crystal structure of an Omega-class glutathione transferase of Bombyx mori, bmGSTO, to gain insight into its catalytic mechanism. The structure of bmGSTO complexed with glutathione determined at a resolution of 2.5Å reveals that it exists as a dimer and is structurally similar to Omega-class GSTs with respect to its secondary and tertiary structures. Analysis of a complex between bmGSTO and glutathione showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTO mutants indicated that amino acid residues Leu62, Lys65, Lys77, Val78, Glu91 and Ser92 in the G-site contribute to catalytic activity.

  9. Structural characterization of the catalytic site of a Nilaparvata lugens delta-class glutathione transferase.

    PubMed

    Yamamoto, Kohji; Higashiura, Akifumi; Hossain, Md Tofazzal; Yamada, Naotaka; Shiotsuki, Takahiro; Nakagawa, Atsushi

    2015-01-15

    Glutathione transferases (GSTs) are a major class of detoxification enzymes that play a central role in the defense against environmental toxicants and oxidative stress. Here, we studied the crystal structure of a delta-class glutathione transferase from Nilaparvata lugens, nlGSTD, to gain insights into its catalytic mechanism. The structure of nlGSTD in complex with glutathione, determined at a resolution of 1.7Å, revealed that it exists as a dimer and its secondary and tertiary structures are similar to those of other delta-class GSTs. Analysis of a complex between nlGSTD and glutathione showed that the bound glutathione was localized to the glutathione-binding site. Site-directed mutagenesis of nlGSTD mutants indicated that amino acid residues Ser11, His52, Glu66, and Phe119 contribute to catalytic activity.

  10. 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

  11. 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

  12. 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

  13. Structural insight into the active site of a Bombyx mori unclassified glutathione transferase.

    PubMed

    Hossain, Md Tofazzal; Yamamoto, Kohji

    2015-01-01

    Glutathione transferases (GSTs) are major detoxification enzymes that play central roles in the defense against various environmental toxicants as well as oxidative stress. Here, we identify amino acid residues of an unclassified GST from Bombyx mori, bmGSTu-interacting glutathione (GSH). Site-directed mutagenesis of bmGSTu mutants indicated that amino acid residues Asp103, Ser162, and Ser166 contribute to catalytic activity.

  14. Anti-peptidyl transferase leader peptides of attenuation-regulated chloramphenicol-resistance genes.

    PubMed Central

    Gu, Z; Harrod, R; Rogers, E J; Lovett, P S

    1994-01-01

    The chloramphenicol (Cm)-inducible cmlA gene of Tn1696 specifies nonenzymatic resistance to Cm and is regulated by attenuation. The first eight codons of the leader specify a peptide that inhibits peptidyl transferase in vitro. Functionally similar, but less inhibitory, peptides are encoded by the leaders of Cm-inducible cat genes. However, the cat and cmlA coding sequences are unrelated and specify proteins of unrelated function. The inhibition of peptidyl transferase by the leader peptides is additive with that of Cm. Erythromycin competes with the inhibitory action of the peptides, and erythromycin and the peptides footprint to overlapping sites at the peptidyl transferase center of 23S rRNA. It is proposed that translation of the cmlA and cat leaders transiently pauses upon synthesis of the inhibitor peptides. The predicted site of pausing is identical to the leader site where long-term occupancy by a ribosome (ribosome stalling) will activate downstream gene expression. We therefore propose the inducer, Cm, converts a peptide-paused ribosome to the stalled state. We discuss the idea that cooperativity between leader peptide and inducer is necessary for ribosome stalling and may link the activation of a specific drug-resistance gene with a particular antibiotic. Images PMID:7515506

  15. 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.

  16. Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes.

    PubMed

    Kalinina, E V; Chernov, N N; Novichkova, M D

    2014-12-01

    Over the last decade fundamentally new features have been revealed for the participation of glutathione and glutathione-dependent enzymes (glutathione transferase and glutaredoxin) in cell proliferation, apoptosis, protein folding, and cell signaling. Reduced glutathione (GSH) plays an important role in maintaining cellular redox status by participating in thiol-disulfide exchange, which regulates a number of cell functions including gene expression and the activity of individual enzymes and enzyme systems. Maintaining optimum GSH/GSSG ratio is essential to cell viability. Decrease in the ratio can serve as an indicator of damage to the cell redox status and of changes in redox-dependent gene regulation. Disturbance of intracellular GSH balance is observed in a number of pathologies including cancer. Consequences of inappropriate GSH/GSSG ratio include significant changes in the mechanism of cellular redox-dependent signaling controlled both nonenzymatically and enzymatically with the participation of isoforms of glutathione transferase and glutaredoxin. This review summarizes recent data on the role of glutathione, glutathione transferase, and glutaredoxin in the regulation of cellular redox-dependent processes.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. Inherited glutathione-S-transferase deficiency is a risk factor for pulmonary asbestosis.

    PubMed

    Smith, C M; Kelsey, K T; Wiencke, J K; Leyden, K; Levin, S; Christiani, D C

    1994-09-01

    Pulmonary diseases attributable to asbestos exposure constitute a significant public health burden, yet few studies have investigated potential genetic determinants of susceptibility to asbestos-related diseases. The glutathione-S-transferases are a family of conjugating enzymes that both catalyze the detoxification of a variety of potentially cytotoxic electrophilic agents and act in the generation of sulfadipeptide leukotriene inflammatory mediators. The gene encoding glutathione-S-transferase class mu (GSTM-1) is polymorphic; approximately 50% of Caucasian individuals have a homozygous deletion of this gene and do not produce functional enzyme. Glutathione-S-transferase mu (GST-mu) deficiency has been previously reported to be associated with smoking-induced lung cancer. We conducted a cross-sectional study to examine the prevalence of the homozygous deletion for the GSTM-1 gene in members of the carpentry trade occupationally exposed to asbestos. Members of the United Brotherhood of Carpenters and Joiners of America attending their 1991 National Union conference were invited to participate. Each participant was offered a chest X-ray and was asked to complete a comprehensive questionnaire and have their blood drawn. All radiographs were assessed for the presence of pneumoconiosis in a blinded fashion by a National Institute for Occupational Safety and Health-certified International Labor Office "B" reader. Individual GSTM-1 status was determined using polymerase chain reaction methods. Six hundred fifty-eight workers were studied. Of these, 80 (12.2%) had X-ray abnormalities associated with asbestos exposure. Individuals genetically deficient in GST-mu were significantly more likely to have radiographic evidence of nonmalignant asbestos-related disease than those who were not deficient (chi 2 = 5.0; P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Bisubstrate UDP-peptide conjugates as human O-GlcNAc transferase inhibitors.

    PubMed

    Borodkin, Vladimir S; Schimpl, Marianne; Gundogdu, Mehmet; Rafie, Karim; Dorfmueller, Helge C; Robinson, David A; van Aalten, Daan M F

    2014-02-01

    Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C₃ linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.

  5. Bisubstrate UDP–peptide conjugates as human O-GlcNAc transferase inhibitors

    PubMed Central

    Borodkin, Vladimir S.; Schimpl, Marianne; Gundogdu, Mehmet; Rafie, Karim; Dorfmueller, Helge C.; Robinson, David A.; vanAalten, Daan M. F.

    2013-01-01

    Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates. PMID:24256146

  6. Genetic Variations in Human Glutathione Transferase Enzymes: Significance for Pharmacology and Toxicology

    PubMed Central

    Josephy, P. David

    2010-01-01

    Glutathione transferase enzymes (GSTs) catalyze reactions in which electrophiles are conjugated to the tripeptide thiol glutathione. While many GST-catalyzed transformations result in the detoxication of xenobiotics, a few substrates, such as dihaloalkanes, undergo bioactivation to reactive intermediates. Many molecular epidemiological studies have tested associations between polymorphisms (especially, deletions) of human GST genes and disease susceptibility or response to therapy. This review presents a discussion of the biochemistry of GSTs, the sources—both genetic and environmental—of interindividual variation in GST activities, and their implications for pharmaco- and toxicogenetics; particular attention is paid to the Theta class GSTs. PMID:20981235

  7. 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.

  8. 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.

  9. Glutathione-binding site of a bombyx mori theta-class glutathione transferase.

    PubMed

    Hossain, M D Tofazzal; Yamada, Naotaka; Yamamoto, Kohji

    2014-01-01

    The glutathione transferase (GST) superfamily plays key roles in the detoxification of various xenobiotics. Here, we report the isolation and characterization of a silkworm protein belonging to a previously reported theta-class GST family. The enzyme (bmGSTT) catalyzes the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)-propane, and 4-nitrophenethyl bromide. Mutagenesis of highly conserved residues in the catalytic site revealed that Glu66 and Ser67 are important for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTT and into the metabolism of exogenous chemical agents.

  10. 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

  11. 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

  12. Binding properties of ferrocene-glutathione conjugates as inhibitors and sensors for glutathione S-transferases.

    PubMed

    Martos-Maldonado, Manuel C; Casas-Solvas, Juan M; Téllez-Sanz, Ramiro; Mesa-Valle, Concepción; Quesada-Soriano, Indalecio; García-Maroto, Federico; Vargas-Berenguel, Antonio; García-Fuentes, Luís

    2012-02-01

    The binding properties of two electroactive glutathione-ferrocene conjugates that consist in glutathione attached to one or both of the cyclopentadienyl rings of ferrocene (GSFc and GSFcSG), to Schistosoma japonica glutathione S-transferase (SjGST) were studied by spectroscopy fluorescence, isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Such ferrocene conjugates resulted to be competitive inhibitors of glutathione S-transferase with an increased binding affinity relative to the natural substrate glutathione (GSH). We found that the conjugate having two glutathione units (GSFcSG) exhibits an affinity for SjGST approximately two orders of magnitude higher than GSH. Furthermore, it shows negative cooperativity with the affinity for the second binding site two orders of magnitude lower than that for the first one. We propose that the reason for such negative cooperativity is steric since, i) the obtained thermodynamic parameters do not indicate profound conformational changes upon GSFcSG binding and ii) docking studies have shown that, when bound, part of the first bound ligand invades the second site due to its large size. In addition, voltammetric measurements show a strong decrease of the peak current upon binding of ferrocene-glutathione conjugates to SjGST and provide very similar K values than those obtained by ITC. Moreover, the sensing ability, expressed by the sensitivity parameter shows that GSFcSG is much more sensitive than GSFc, for the detection of SjGST.

  13. Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases

    PubMed Central

    He, Weizhi; Zhang, Xuhui; Zhang, Jun; Jia, Xu; Zhang, Jing; Sun, Wenxia; Jiang, Hengyi; Chen, Dongrong; Murchie, Alastair IH

    2013-01-01

    The acquisition of antibiotic resistance by human pathogens poses a significant threat to public health. The mechanisms that control the proliferation and expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are a historically important class of antibiotics that were introduced in the 1940s. Aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug or enzymatic modification of the target rRNA through methylation or through the overexpression of efflux pumps. In our recent paper, we reported that expression of the aminoglycoside resistance genes encoding the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes was controlled by an aminoglycoside-sensing riboswitch RNA. This riboswitch is embedded in the leader RNA of the aac/aad genes and is associated with the integron cassette system. The leader RNA can sense and bind specific aminoglycosides such that the binding causes a structural transition in the leader RNA, which leads to the induction of aminoglycoside antibiotic resistance. Specific aminoglycosides induce reporter gene expression mediated by the leader RNA. Aminoglycoside RNA binding was measured directly and, aminoglycoside-induced changes in RNA structure monitored by chemical probing. UV cross-linking and mutational analysis identified potential aminoglycoside binding sites on the RNA. PMID:23880830

  14. 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

  15. Identification of Glutathione S-Transferase Pi as a Protein Involved in Parkinson Disease Progression

    PubMed Central

    Shi, Min; Bradner, Joshua; Bammler, Theo K.; Eaton, David L.; Zhang, JianPeng; Ye, ZuCheng; Wilson, Angela M.; Montine, Thomas J.; Pan, Catherine; Zhang, Jing

    2009-01-01

    Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD. PMID:19498008

  16. 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.

  17. 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

  18. Miners compensated for pneumoconiosis and glutathione s-transferases M1 and T1 genotypes.

    PubMed

    Zimmermann, Anna; Ebbinghaus, Rainer; Prager, Hans-Martin; Blaszkewicz, Meinolf; Hengstler, Jan G; Golka, Klaus

    2012-01-01

    Chronic inhalation of quartz-containing dust produces reversible inflammatory changes in lungs resulting in irreversible fibrotic changes termed pneumoconiosis. Due to the inflammatory process in the lungs, highly reactive substances are released that may be detoxified by glutathione S-transferases. Therefore, 90 hard coal miners with pneumoconiosis as a recognized occupational disease (in Germany: Berufskrankheit BK 4101) were genotyped for glutathione S-transferases M1 (GSTM1) and T1 (GSTT1) according to standard methods. Furthermore, occupational exposure and smoking habits were assessed by questionnaire. Changes in a chest x-ray were classified according to ILO classification 2000. Of the investigated hard coal miners 43% were GSTM1 negative whereas 57% were GSTM1 positive. The arithmetic mean of the age at time of investigation was 74.2 yr (range: 42-87 yr). Seventy-four percent of the hard coal miners reported being ever smokers, while 26% denied smoking. All hard coal miners provided pneumoconiosis-related changes in the chest x-ray. The observed frequency of GSTM1 negative hard coal miners was not different from frequencies reported for general Caucasian populations and in agreement with findings reported for Chinese coal miners. In contrast, in a former study, 16 of 19 German hard coal miners (84%) with urinary bladder cancer displayed a GSTM1 negative genotype. The outcome of this study provides evidence that severely occupationally exposed Caucasian hard coal miners do not present an elevated level of GSTM1 negative individuals. PMID:22686319

  19. 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

  20. 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

  1. 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

  2. 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

  3. SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center

    PubMed Central

    Bhushan, Shashi; Hoffmann, Thomas; Seidelt, Birgit; Frauenfeld, Jens; Mielke, Thorsten; Berninghausen, Otto; Wilson, Daniel N.; Beckmann, Roland

    2011-01-01

    As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNAPro in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide. PMID:21267063

  4. Lectin domains of polypeptide GalNAc transferases exhibit glycopeptide binding specificity.

    PubMed

    Pedersen, Johannes W; Bennett, Eric P; Schjoldager, Katrine T-B G; Meldal, Morten; Holmér, Andreas P; Blixt, Ola; Cló, Emiliano; Levery, Steven B; Clausen, Henrik; Wandall, Hans H

    2011-09-16

    UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence specificity, whereas the primary function of the lectin domain is to increase affinity to previously glycosylated substrates. Whether the lectin domain also has peptide sequence selectivity has remained unclear. Using a glycopeptide array with a library of synthetic and recombinant glycopeptides based on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate an additional level of complexity in the initiation step of O-glycosylation by GalNAc-Ts.

  5. 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

  6. Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center

    PubMed Central

    Marks, James; Kannan, Krishna; Roncase, Emily J.; Klepacki, Dorota; Kefi, Amira; Orelle, Cédric; Vázquez-Laslop, Nora; Mankin, Alexander S.

    2016-01-01

    The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center. PMID:27791002

  7. High yield production of myristoylated Arf6 small GTPase by recombinant N-myristoyl transferase

    PubMed Central

    Padovani, Dominique; Zeghouf, Mahel; Traverso, José A.; Giglione, Carmela; Cherfils, Jacqueline

    2013-01-01

    Small GTP-binding proteins of the Arf family (Arf GTPases) interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Understanding the underlying molecular mechanisms requires in vitro biochemical assays to test for regulations and functions. Such assays should use proteins in their cellular form, which carry a myristoyl lipid attached in N-terminus. N-myristoylation of recombinant Arf GTPases can be achieved by co-expression in E. coli with a eukaryotic N-myristoyl transferase. However, purifying myristoylated Arf GTPases is difficult and has a poor overall yield. Here we show that human Arf6 can be N-myristoylated in vitro by recombinant N-myristoyl transferases from different eukaryotic species. The catalytic efficiency depended strongly on the guanine nucleotide state and was highest for Arf6-GTP. Large-scale production of highly pure N-myristoylated Arf6 could be achieved, which was fully functional for liposome-binding and EFA6-stimulated nucleotide exchange assays. This establishes in vitro myristoylation as a novel and simple method that could be used to produce other myristoylated Arf and Arf-like GTPases for biochemical assays. PMID:23319116

  8. Predicted binding of certain antifilarial compounds with glutathione-S-transferase of human Filariids

    PubMed Central

    Saeed, Mohd; Baig, Mohd Hassan; Bajpai, Preeti; Srivastava, Ashwini Kumar; Ahmad, Khurshid; Mustafa, Huma

    2013-01-01

    Glutathione-S-transferase is a major phase-II detoxification enzyme in parasitic helminthes. Previous research highlights the importance of GSTs in the establishment of chronic infections in cytotoxic microenvironments. Filarial nematodes depend on these detoxification enzymes for their survival in the host. GST plays an important role in filariasis and other diseases. GST from W.bancrofti and B.malayi are very much different from human GST. This structural difference makes GST potential chemotherapeutic targets for antifilarial treatment. In this study we have checked the efficacy of some well known antifilarial compounds against GST from B.malayi and W.bancrofti. The structure of BmGST was modeled using modeller9v10 and was submitted to PMDB. Molecular docking study reveals arbindazole to be the most potent compounds against GST from both the filarial parasites. Role of some residues playing important role in the binding of compounds within the active site of GST has also been revealed in the present study. The BmGST and WbGST structural information and docking studies could aid in screening new antifilarials or selective inhibitors for chemotherapy against filariasis. Abbreviations GST - Glutathione-S-transferase, Bm - Brugia malayi, Wb - Wuchereria bancrofti. PMID:23516334

  9. 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

  10. Conductimetric assays for the hydrolase and transferase activities of phospholipase D enzymes.

    PubMed

    Mezna, M; Lawrence, A J

    1994-05-01

    Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.

  11. Corneal aldehyde dehydrogenase and glutathione S-transferase activity after excimer laser keratectomy in guinea pigs

    PubMed Central

    Bilgihan, K.; Bilgihan, A.; Turkozkan, N.

    1998-01-01

    BACKGROUND—The free radical balance of the eye may be changed by excimer laser keratectomy. Previous studies have demonstrated that excimer laser keratectomy increases the corneal temperature, decreases the superoxide dismutase activity of the aqueous, and induces lipid peroxidation in the superficial corneal stroma. Aldehyde dehydrogenase (ALDH) and glutathione S-transferase (GST) are known to play an important role in corneal metabolism, particularly in detoxification of aldehydes, which are generated from free radical reactions.
METHODS—In three groups of guinea pigs mechanical corneal de-epithelialisation was performed in group I, superficial corneal photoablation in group II, and deep corneal photoablation in group III, and the corneal ALDH and GST activities measured after 48 hours.
RESULTS—The mean ALDH and GST activities of group I and II showed no differences compared with the controls (p>0.05). The corneal ALDH activities were found to be significantly decreased (p<0.05) and GST activities increased (p<0.05) in group III.
CONCLUSION—These results suggest that excimer laser treatment of high myopia may change the ALDH and GST activities, metabolism, and free radical balance of the cornea.

 Keywords: excimer laser keratectomy; aldehyde dehydrogenase; glutathione S-transferase PMID:9602629

  12. Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris.

    PubMed

    Chronopoulou, Evangelia; Madesis, Panagiotis; Asimakopoulou, Basiliki; Platis, Dimitrios; Tsaftaris, Athanasios; Labrou, Nikolaos E

    2012-06-01

    Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.

  13. Substrate profiling of glutathione S-transferase with engineered enzymes and matched glutathione analogues.

    PubMed

    Feng, Shan; Zhang, Lei; Adilijiang, Gulishana; Liu, Jieyuan; Luo, Minkui; Deng, Haiteng

    2014-07-01

    The identification of specific substrates of glutathione S-transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G-W40A-R41A mutant (GST-KWR) of the mu-class glutathione S-transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH-R1) as the cosubstrate. The GSH-R1 conjugation products can react with an azido-based biotin probe for ready enrichment and MS identification. Proof-of-principle studies were carried to detect the products of GSH-R1 conjugation to 1-chloro-2,4-dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.

  14. Evolutionary divergence of Ure2pA glutathione transferases in wood degrading fungi.

    PubMed

    Roret, Thomas; Thuillier, Anne; Favier, Frédérique; Gelhaye, Eric; Didierjean, Claude; Morel-Rouhier, Mélanie

    2015-10-01

    The intracellular systems of detoxification are crucial for the survival of wood degrading fungi. Within these systems, glutathione transferases could play a major role since this family of enzymes is specifically extended in lignolytic fungi. In particular the Ure2p class represents one third of the total GST number in Phanerochaete chrysosporium. These proteins have been phylogenetically split into two subclasses called Ure2pA and Ure2pB. Ure2pB can be classified as Nu GSTs because of shared structural and functional features with previously characterized bacterial isoforms. Ure2pA can rather be qualified as Nu-like GSTs since they exhibit a number of differences. Ure2pA possess a classical transferase activity, a more divergent catalytic site and a higher structural flexibility for some of them, compared to Nu GSTs. The characterization of four members of this Ure2pA subclass (PcUre2pA4, PcUre2pA5, PcUre2pA6 and PcUre2pA8) revealed specific functional and structural features, suggesting that these enzymes have rapidly evolved and differentiated, probably to adapt to the complex chemical environment associated with wood decomposition.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Purification and partial characterization of glutathione transferase from the teleost Monopterus albus.

    PubMed

    Huang, Qing; Liang, Li; Wei, Tao; Zhang, Daming; Zeng, Qing-Yin

    2008-01-01

    Glutathione transferases (GSTs) catalyze the transfer of glutathione to a variety of xenobiotic and toxic endogenous compounds. GSTs are phase II biotransformation enzymes and are proposed as biomarkers of environmental pollution. In this study, a cytosolic glutathione transferase (maGST) was purified from liver of the freshwater fish Monopterus albus by affinity chromatography. The maGST appeared to be a homodimer composed of two subunits each with a molecular weight of 26 kDa. This maGST showed high activity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with CDNB as substrate revealed a K(m) of 0.28 mM and V(max) of 15.68 micromol/min per mg of protein. It had maximum activity in the pH range 7.0-7.5, a broad optimum T(m) range of 30 degrees C-55 degrees C, and a high thermal stability with 77% of its initial activity at 45 degrees C. This high thermal stability of maGST could be related to the physiological adaptation of M. albus to high temperatures in tropical and subtropical environments.

  20. Miners compensated for pneumoconiosis and glutathione s-transferases M1 and T1 genotypes.

    PubMed

    Zimmermann, Anna; Ebbinghaus, Rainer; Prager, Hans-Martin; Blaszkewicz, Meinolf; Hengstler, Jan G; Golka, Klaus

    2012-01-01

    Chronic inhalation of quartz-containing dust produces reversible inflammatory changes in lungs resulting in irreversible fibrotic changes termed pneumoconiosis. Due to the inflammatory process in the lungs, highly reactive substances are released that may be detoxified by glutathione S-transferases. Therefore, 90 hard coal miners with pneumoconiosis as a recognized occupational disease (in Germany: Berufskrankheit BK 4101) were genotyped for glutathione S-transferases M1 (GSTM1) and T1 (GSTT1) according to standard methods. Furthermore, occupational exposure and smoking habits were assessed by questionnaire. Changes in a chest x-ray were classified according to ILO classification 2000. Of the investigated hard coal miners 43% were GSTM1 negative whereas 57% were GSTM1 positive. The arithmetic mean of the age at time of investigation was 74.2 yr (range: 42-87 yr). Seventy-four percent of the hard coal miners reported being ever smokers, while 26% denied smoking. All hard coal miners provided pneumoconiosis-related changes in the chest x-ray. The observed frequency of GSTM1 negative hard coal miners was not different from frequencies reported for general Caucasian populations and in agreement with findings reported for Chinese coal miners. In contrast, in a former study, 16 of 19 German hard coal miners (84%) with urinary bladder cancer displayed a GSTM1 negative genotype. The outcome of this study provides evidence that severely occupationally exposed Caucasian hard coal miners do not present an elevated level of GSTM1 negative individuals.

  1. Indication for joint replacement and glutathione s-transferases M1 and T1 genotypes.

    PubMed

    Klein, Torsten; Selinski, Silvia; Blaszkewicz, Meinolf; Hengstler, Jan G; Golka, Klaus

    2012-01-01

    In most patients with osteoarthritis (OA), therapy-resistant pain is the indication for hip or knee replacement. Glutathione S-transferases, particularly glutathione S-transferase M1 (GSTM1), are involved in metabolism of highly reactive metabolites that may be generated by inflammatory processes. In total, 148 patients with indication for hip or knee replacement and 129 patients of the same hospital without indication for joint replacement were genotyped for GSTM1 and GSTT1 and interviewed by a newly developed questionnaire for occupational and nonoccupational risk factors of hip and/or knee osteoarthritis. Mean age was 70.9 yr in OA cases and 67.4 yr in controls. The frequency of GSTM1 negative in the OA case group was (45%) in the lower range compared to values in Caucasian general population (approximately 50%), whereas the frequency in the controls was normal (51%). The frequency of GSTT1 negative genotype in OA cases and controls was normal. The normal distribution of the GSTM1 negative genotype in patients with indication for hip or knee replacement indicates that the role GSTM1 in these patients is different from that in other aseptic inflammatory diseases such as ozone-related inflammatory reactions of the respiratory tract.

  2. 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

  3. Mice Deficient in Glutathione Transferase Zeta/Maleylacetoacetate Isomerase Exhibit a Range of Pathological Changes and Elevated Expression of Alpha, Mu, and Pi Class Glutathione Transferases

    PubMed Central

    Lim, Cindy E.L.; Matthaei, Klaus I.; Blackburn, Anneke C.; Davis, Richard P.; Dahlstrom, Jane E.; Koina, Mark E.; Anders, M.W.; Board, Philip G.

    2004-01-01

    Glutathione transferase zeta (GSTZ1-1) is the major enzyme that catalyzes the metabolism of α-halo acids such as dichloroacetic acid, a carcinogenic contaminant of chlorinated water. GSTZ1-1 is identical with maleylacetoacetate isomerase, which catalyzes the penultimate step in the catabolic pathways for phenylalanine and tyrosine. In this study we have deleted the Gstz1 gene in BALB/c mice and characterized their phenotype. Gstz1−/− mice do not have demonstrable activity with maleylacetone and α-halo acid substrates, and other GSTs do not compensate for the loss of this enzyme. When fed a standard diet, the GSTZ1-1-deficient mice showed enlarged liver and kidneys as well as splenic atrophy. Light and electron microscopic examination revealed multifocal hepatitis and ultrastructural changes in the kidney. The addition of 3% (w/v) phenylalanine to the drinking water was lethal for young mice (<28 days old) and caused liver necrosis, macrovesicular steatosis, splenic atrophy, and a significant loss of circulating leukocytes in older surviving mice. GSTZ1-1-deficient mice showed constitutive induction of alpha, mu, and pi class GSTs as well as NAD(P)H:quinone oxidoreductase 1. The overall response is consistent with the chronic accumulation of a toxic metabolite(s). We detected the accumulation of succinylacetone in the serum of deficient mice but cannot exclude the possibility that maleylacetoacetate and maleylacetone may also accumulate. PMID:15277241

  4. 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.

  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. 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

  7. Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical

    PubMed Central

    Kumari, Vandana; Dyba, Marzena A.; Holland, Ryan J.; Liang, Yu-He; Singh, Shivendra V.

    2016-01-01

    Dietary isothiocyanates abundant as glucosinolate precursors in many edible cruciferous vegetables are effective for prevention of cancer in chemically-induced and transgenic rodent models. Some of these agents, including phenethyl isothiocyanate (PEITC), have already advanced to clinical investigations. The primary route of isothiocyanate metabolism is its conjugation with glutathione (GSH), a reaction catalyzed by glutathione S-transferase (GST). The pi class GST of subunit type 1 (hGSTP1) is much more effective than the alpha class GST of subunit type 1 (hGSTA1) in catalyzing the conjugation. Here, we report the crystal structures of hGSTP1 and hGSTA1 each in complex with the GSH adduct of PEITC. We find that PEITC also covalently modifies the cysteine side chains of GST, which irreversibly inhibits enzymatic activity. PMID:27684484

  8. 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.

  9. 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.

  10. Diversification of Fungal Specific Class A Glutathione Transferases in Saprotrophic Fungi

    PubMed Central

    Favier, Frédérique; Harvengt, Luc; Didierjean, Claude; Jacquot, Jean-Pierre; Morel-Rouhier, Mélanie; Gelhaye, Eric

    2013-01-01

    Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes and endogenous metabolism. The distribution of fungal-specific class A GSTs was investigated in saprotrophic fungi revealing a recent diversification within this class. Biochemical characterization of eight GSTFuA isoforms from Phanerochaete chrysosporium and Coprinus cinereus demonstrated functional diversity in saprotrophic fungi. The three-dimensional structures of three P. chrysosporium isoforms feature structural differences explaining the functional diversity of these enzymes. Competition experiments between fluorescent probes, and various molecules, showed that these GSTs function as ligandins with various small aromatic compounds, derived from lignin degradation or not, at a L-site overlapping the glutathione binding pocket. By combining genomic data with structural and biochemical determinations, we propose that this class of GST has evolved in response to environmental constraints induced by wood chemistry. PMID:24278272

  11. Molybdenum and tungsten oxygen transferases--and functional diversity within a common active site motif.

    PubMed

    Pushie, M Jake; Cotelesage, Julien J; George, Graham N

    2014-01-01

    Molybdenum and tungsten are the only second and third-row transition elements with a known function in living organisms. The molybdenum and tungsten enzymes show common structural features, with the metal being bound by a pyranopterin-dithiolene cofactor called molybdopterin. They catalyze a variety of oxygen transferase reactions coupled with two-electron redox chemistry in which the metal cycles between the +6 and +4 oxidation states usually with water, either product or substrate, providing the oxygen. The functional roles filled by the molybdenum and tungsten enzymes are diverse; for example, they play essential roles in microbial respiration, in the uptake of nitrogen in green plants, and in human health. Together, the enzymes form a superfamily which is among the most prevalent known, being found in all kingdoms of life. This review discusses what is known of the active site structures and the mechanisms, together with some recent insights into the evolution of these important enzyme systems.

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

    PubMed

    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.

  13. 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.

  14. Atypical features of a Ure2p glutathione transferase from Phanerochaete chrysosporium.

    PubMed

    Thuillier, Anne; Roret, Thomas; Favier, Frédérique; Gelhaye, Eric; Jacquot, Jean-Pierre; Didierjean, Claude; Morel-Rouhier, Mélanie

    2013-07-11

    Glutathione transferases (GSTs) are known to transfer glutathione onto small hydrophobic molecules in detoxification reactions. The GST Ure2pB1 from Phanerochaete chrysosporium exhibits atypical features, i.e. the presence of two glutathione binding sites and a high affinity towards oxidized glutathione. Moreover, PcUre2pB1 is able to efficiently deglutathionylate GS-phenacylacetophenone. Catalysis is not mediated by the cysteines of the protein but rather by the one of glutathione and an asparagine residue plays a key role in glutathione stabilization. Interestingly PcUre2pB1 interacts in vitro with a GST of the omega class. These properties are discussed in the physiological context of wood degrading fungi.

  15. 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.

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

    PubMed

    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.

  17. 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

  18. Probing functions of the ribosomal peptidyl transferase center by nucleotide analog interference.

    PubMed

    Erlacher, Matthias D; Polacek, Norbert

    2012-01-01

    The ribosome is a huge ribonucleoprotein complex in charge of protein synthesis in every living cell. The catalytic center of this dynamic molecular machine is entirely built up of 23S ribosomal RNA and therefore the ribosome can be referred to as the largest natural ribozyme known so far. The in vitro reconstitution approach of large ribosomal subunits described herein allows nucleotide analog interference studies to be performed. The approach is based on the site-specific introduction of nonnatural nucleotide analogs into the peptidyl transferase center, the active site located on the interface side of the large ribosomal subunit. This method combined with standard tests of ribosomal functions broadens the biochemical repertoire to investigate the mechanism of diverse aspects of translation considerably and adds another layer of molecular information on top of structural and mutational studies of the ribosome. PMID:22315072

  19. 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

  20. Crystal structure of Glycine max glutathione transferase in complex with glutathione: investigation of the mechanism operating by the Tau class glutathione transferases.

    PubMed

    Axarli, Irene; Dhavala, Prathusha; Papageorgiou, Anastassios C; Labrou, Nikolaos E

    2009-08-13

    Cytosolic GSTs (glutathione transferases) are a multifunctional group of enzymes widely distributed in Nature and involved in cellular detoxification processes. The three-dimensional structure of GmGSTU4-4 (Glycine max GST Tau 4-4) complexed with GSH was determined by the molecular replacement method at 2.7 A (1 A=0.1 nm) resolution. The bound GSH is located in a region formed by the beginning of alpha-helices H1, H2 and H3 in the N-terminal domain of the enzyme. Significant differences in the G-site (GSH-binding site) as compared with the structure determined in complex with Nb-GSH [S-(p-nitrobenzyl)-glutathione] were found. These differences were identified in the hydrogen-bonding and electrostatic interaction pattern and, consequently, GSH was found bound in two different conformations. In one subunit, the enzyme forms a complex with the ionized form of GSH, whereas in the other subunit it can form a complex with the non-ionized form. However, only the ionized form of GSH may form a productive and catalytically competent complex. Furthermore, a comparison of the GSH-bound structure with the Nb-GSH-bound structure shows a significant movement of the upper part of alpha-helix H4 and the C-terminal. This indicates an intrasubunit modulation between the G-site and the H-site (electrophile-binding site), suggesting that the enzyme recognizes the xenobiotic substrates by an induced-fit mechanism. The reorganization of Arg111 and Tyr107 upon xenobiotic substrate binding appears to govern the intrasubunit structural communication between the G- and H-site and the binding of GSH. The structural observations were further verified by steady-state kinetic analysis and site-directed mutagenesis studies.

  1. Crystal structures of Acetobacter aceti succinyl-coenzyme A (CoA):acetate CoA-transferase reveal specificity determinants and illustrate the mechanism used by class I CoA-transferases.

    PubMed

    Mullins, Elwood A; Kappock, T Joseph

    2012-10-23

    Coenzyme A (CoA)-transferases catalyze transthioesterification reactions involving acyl-CoA substrates, using an active-site carboxylate to form covalent acyl anhydride and CoA thioester adducts. Mechanistic studies of class I CoA-transferases suggested that acyl-CoA binding energy is used to accelerate rate-limiting acyl transfers by compressing the substrate thioester tightly against the catalytic glutamate [White, H., and Jencks, W. P. (1976) J. Biol. Chem. 251, 1688-1699]. The class I CoA-transferase succinyl-CoA:acetate CoA-transferase is an acetic acid resistance factor (AarC) with a role in a variant citric acid cycle in Acetobacter aceti. In an effort to identify residues involved in substrate recognition, X-ray crystal structures of a C-terminally His(6)-tagged form (AarCH6) were determined for several wild-type and mutant complexes, including freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts. The latter shows the acetate product bound to an auxiliary site that is required for efficient carboxylate substrate recognition. A mutant in which the catalytic glutamate was changed to an alanine crystallized in a closed complex containing dethiaacetyl-CoA, which adopts an unusual curled conformation. A model of the acetyl-CoA Michaelis complex demonstrates the compression anticipated four decades ago by Jencks and reveals that the nucleophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of Gly388 NH and CoA N2″. CoA is nearly immobile along its entire length during all stages of the enzyme reaction. Spatial and sequence conservation of key residues indicates that this mechanism is general among class I CoA-transferases.

  2. Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids

    SciTech Connect

    Wiesenborn, D.P.; Rudolph, F.B.; Papoutsakis, E.T. )

    1989-02-01

    Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the K{sub m} values at pH 7.5 and 30{degree}C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the K{sub m} value for acetoacetyl-CoA ranged from about 7 to 56{mu}M, depending on the acid substrate. The K{sub m} values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

  3. RMF inactivates ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel.

    PubMed

    Yoshida, Hideji; Yamamoto, Hiroshi; Uchiumi, Toshio; Wada, Akira

    2004-04-01

    In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S ribosomes under stress conditions. The 100S ribosome, a dimer of 70S ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S ribosomes. During the stationary phase, most of the 70S ribosomes turn to 100S ribosomes, which have lost translational activity. This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase. If stationary phase cells are transferred to fresh medium, the 100S ribosomes immediately go back to active 70S ribosomes, showing that inactive 100S <--> active 70S interconversion is a major system regulating translation activity in stationary phase cells. To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E. coli were examined by a chemical probing method using dimethyl sulphate (DMS). As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced. Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity. We conclude that RMF inactivates ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel. It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.

  4. pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

    PubMed Central

    Muth, G W; Chen, L; Kosek, A B; Strobel, S A

    2001-01-01

    A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known. PMID:11680845

  5. Glutathione S-transferase isoenzymes in human tumours and tumour derived cell lines.

    PubMed Central

    Lewis, A. D.; Forrester, L. M.; Hayes, J. D.; Wareing, C. J.; Carmichael, J.; Harris, A. L.; Mooghen, M.; Wolf, C. R.

    1989-01-01

    An increasing body of evidence indicates that glutathione S-transferases play a role in the intrinsic and acquired resistance of tumours to anticancer drugs. In view of the wide use of tumour cell lines to understand the factors which confer either sensitivity or resistance to chemotherapeutic agents we have determined glutathione S-transferase (GST) activity and isozyme composition in nine human cell lines. These data have been compared with the values obtained in solid tumours. In most cases overall GST activity was higher in the tumours than in the cell lines. This was most pronounced for the breast tumour samples relative to MCF7 cell line. The pi class GST subunit was present at similar concentration in the cell lines and the tumours, and in most cases was the most abundant subunit present. The alpha and mu class GST were expressed in most of the cell lines but at much lower concentration than the pi class subunit. Also considerable variability particularly in the expression of the mu subunits was observed. This was also the case for the expression of these subunits in the solid tumour samples. The levels of these GSTs (when expressed) in the solid tumours was invariably higher than that observed in the cell lines. There are therefore several similarities but also some significant differences in GST expression in solid tumours and cell lines. Whether the differences are because expression is lost during the generation of the cell lines or whether it reflects the individuality of human tumours remains to be clearly established. Images Figure 2 Figure 4 PMID:2789940

  6. Glutathione-S-transferase GST M1 "null" genotype and the risk of alcoholic liver disease.

    PubMed

    Savolainen, V T; Pjarinen, J; Perola, M; Penttilä, A; Karhunen, P J

    1996-11-01

    The present study was conducted to investigate possible association between the occurrence of glutathione-S-transferase GST M1 "null" genotype and alcoholic liver disease (ALD). The"null" genotype indicating absent activity of class mu glutathione transferase was assessed in 33 abstainers, 43 moderate alcohol consumers, and 313 heavy alcohol consumers by polymerase chain reaction. The genotypes were compared with occurrence of alcoholic fatty liver, alcoholic hepatitis, and alcoholic liver fibrosis. The "null" genotype was found among 44.7% of patients in the series, with no significant differences between different consumption groups: controls, 36.4%; moderate consumers, 39.5%; and heavy consumers, 46.3%. Occurrence of GST M1 "null" genotype was not associated with occurrence ALD among moderate alcohol consumers. Frequency of the "null" genotype was, however, statistically nearly significantly [p = 0.07, odds ratio (OR) = 1.75] lower among heavy consumers with normal liver histology than in alcoholics with ALD. Furthermore, when compared with heavy consumers without ALD, the "null" genotype was nearly significantly more frequent among heavy consumers with at least slight liver fibrosis (p = 0.05, OR = 1.8) and statistically significantly more frequent among among alcoholics with advanced liver fibrosis (p < 0.025, OR = 2.3). Results of the present Finnish association study suggest that homozygous deletion of the GST M1 gene may indicate increased susceptibility to develop irreversible liver damage in response to the toxic effects of ethanol. Significant association was found between the occurrence of the "null" genotype and the occurrence of alcoholic liver cirrhosis.

  7. A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.

    PubMed

    Skopelitou, Katholiki; Dhavala, Prathusha; Papageorgiou, Anastassios C; Labrou, Nikolaos E

    2012-01-01

    In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity.

  8. Design, synthesis, and evaluation of latent alkylating agents activated by glutathione S-transferase.

    PubMed

    Satyam, A; Hocker, M D; Kane-Maguire, K A; Morgan, A S; Villar, H O; Lyttle, M H

    1996-04-12

    In search of compounds with improved specificity for targeting the important cancer-associated P1-1 glutathione S-transferase (GST) isozyme, new analogs 4 and 5 of the previously reported glutathione S-transferase (GST)-activated latent alkylating agent gamma-glutamyl-alpha-amino-beta-[[[2-[[bis[bis(2-chloroethyl)amino]ph osp horyl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)-phenylglycine (3) have been designed, synthesized, and evaluated. One of the diastereomers of 4 exhibited good selectivity for GST P1-1. The tetrabromo analog 5 of the tetrachloro compound 3 maintained its specificity and was found to be more readily activated by GSTs than 3. The GST activation concept was further broadened through design, synthesis, and evaluation of a novel latent urethane mustard 8 and its diethyl ester 9. Interestingly, 8 showed very good specificity for P1-1 GST. Cell culture studies were carried out on 4, 5, 8, and 9 using cell lines engineered to have varying levels of GST P1-1 isozyme. New analogs 4 and 5 exhibited increased toxicity to cell lines with overexpressed GST P1-1 isozyme. The urethane mustard 8 and its diethyl ester 9 were found to be not as toxic. However, they too exhibited more toxicity to a cell line engineered to have elevated P1-1 levels, which was in agreement with the observed in vitro specificity of 8 for P1-1 GST isozyme. Mechanistic studies on alkaline as well as enzyme-catalyzed decomposition of latent mustard 3 provided experimental proof for the hypothesis that 3 breaks down into an active phosphoramidate mustard and a reactive vinyl sulfone. The alkylating nature of the decomposition products was further demonstrated by trapping those transient species as relatively stable diethyldithiocarbamic acid adducts. These results substantially extend previous efforts to develop drugs targeting GST and provide a paradigm for development of other latent drugs. PMID:8648613

  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. 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

  11. 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...

  12. Function and phylogeny of bacterial butyryl-CoA:acetate transferases and their diversity in the proximal colon of swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studying the host-associated butyrate-producing bacterial community is important because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl-coA:acetate transferase (2.3.8.3) as a the main gene for butyrate production in intestinal ecosystems; h...

  13. CT-GalNAc transferase overexpression in adult mice is associated with extrasynaptic utrophin in skeletal muscle fibres.

    PubMed

    Durko, Margaret; Allen, Carol; Nalbantoglu, Josephine; Karpati, George

    2010-09-01

    Duchenne muscular dystrophy is a genetic muscle disease characterized by the absence of sub-sarcolemmal dystrophin that results in muscle fibre necrosis, progressive muscle wasting and is fatal. Numerous experimental studies with dystrophin-deficient mdx mice, an animal model for the disease, have demonstrated that extrasynaptic upregulation of utrophin, an analogue of dystrophin, can prevent muscle fibre deterioration and reduce or negate the dystrophic phenotype. A different approach for ectopic expression of utrophin relies on augmentation of CT-GalNAc transferase in muscle fibre. We investigated whether CT-GalNAc transferase overexpression in adult mice influence appearance of utrophin in the extrasynaptic sarcolemma. After electrotransfer of plasmid DNA carrying an expression cassette of CT-GalNAc transferase into tibialis anterior muscle of wild type and dystrophic mice, muscle sections were examined by immunofluorescence. CT-GalNAc transgene expression augmented sarcolemmal carbohydrate glycosylation and was accompanied by extrasynaptic utrophin. A 6-week time course study showed that the highest efficiency of utrophin overexpression in a plasmid harboured muscle fibres was 32.2% in CD-1 and 52% in mdx mice, 2 and 4 weeks after CT-GalNAc gene transfer, respectively. The study provides evidence that postnatal CT-GalNAc transferase overexpression stimulates utrophin upregulation that is inherently beneficial for muscle structure and strength restoration. Thus CT-GalNAc may provide an important therapeutic molecule for treatment of dystrophin deficiency in Duchenne muscular dystrophy.

  14. 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...

  15. 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...

  16. Characterization of the hydrophobic substrate-binding site of the bacterial beta class glutathione transferase from Proteus mirabilis.

    PubMed

    Federici, Luca; Masulli, Michele; Di Ilio, Carmine; Allocati, Nerino

    2010-09-01

    Since their discovery, bacterial glutathione (GSH)transferases have been characterized in terms of their ability to catalyse a variety of different reactions on a large set of toxic molecules of xenobiotic or endobiotic origin. Furthermore the contribution of different residues in the GSH-binding site to GSH activation has been extensively investigated. Little is known, however, about the contribution to catalysis and overall stability of single residues shaping the hydrophobic co-substrate binding site (H-site). Here we tackle this problem by site-directed mutagenesis of residues facing the H-site in the bacterial beta class GSH transferase from Proteus mirabilis. We investigate the behaviour of these mutants under a variety of conditions and analyse their activity against several co-substrates, representative of the different reactions catalyzed by bacterial GSH transferases. Our work shows that mutations at the H-site can be used to modulate activity at the level of the different catalytic mechanisms operating on the chosen substrates, each mutation showing a different fingerprint. This work paves the way for future studies aimed at improving the catalytic properties of beta class GSH transferases against selected substrates for bioremediation purposes.

  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. 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...

  19. An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation.

    PubMed

    Fung, Angela W; Ebhardt, H Alexander; Abeysundara, Heshani; Moore, Jack; Xu, Zhizhong; Fahlman, Richard P

    2011-06-17

    Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.

  20. 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

  1. Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity.

    PubMed Central

    Jackson, B J; Bohin, J P; Kennedy, E P

    1984-01-01

    Phosphoglycerol transferase I, an enzyme of the inner, cytoplasmic membrane of Escherichia coli, catalyzes the in vitro transfer of phosphoglycerol residues from phosphatidylglycerol to membrane-derived oligosaccharides or to the model substrate arbutin (p-hydroxyphenyl-beta-D-glucoside). The products are a phosphoglycerol diester derivative of membrane-derived oligosaccharides or arbutin, respectively, and sn-1,2-diglyceride (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983). Because this enzyme has its active site on the outer aspect of the inner membrane, it also catalyzes the transfer of phosphoglycerol residues to arbutin added to the medium (J.-P. Bohin and E. P. Kennedy, J. Biol. Chem. 259:8388-8393, 1984). When strains bearing the dgk mutation, which are defective in the enzyme diglyceride kinase, are grown in medium containing arbutin, they accumulate large amounts of sn-1,2-diglyceride, a product of the phosphoglycerol transferase I reaction. Growth is inhibited under these conditions. A further mutation in such a dgk strain, leading to the loss of phosphoglycerol transferase I activity, should result in the phenotype of arbutin resistance. We have exploited this fact to obtain strains with such mutations, designated mdoB, that map near min 99. Such mutants lack detectable phosphoglycerol transferase I activity, cannot transfer phosphoglycerol residues to arbutin in vivo, and synthesize membrane-derived oligosaccharides devoid of phosphoglycerol residues. These findings offer strong genetic support for the function of phosphoglycerol transferase I in membrane-derived oligosaccharide biosynthesis. PMID:6094515

  2. 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.

  3. 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

  4. 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.

  5. Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates.

    PubMed

    Scott, Deborah J; da Costa, Bernardo M T; Espy, Stephanie C; Keasling, Jay D; Cornish, Katrina

    2003-09-01

    Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP)

  6. Heterologous expression and functional characterization of avian mu-class glutathione S-transferases.

    PubMed

    Bunderson, Brett R; Kim, Ji Eun; Croasdell, Amanda; Mendoza, Kristelle M; Reed, Kent M; Coulombe, Roger A

    2013-08-01

    Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B1 (AFB1), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB1-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB1, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB1 detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA

  7. 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

  8. 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

  9. Joint effect of glutathione S-transferase genotypes and cigarette smoking on idiopathic male infertility.

    PubMed

    Yarosh, S L; Kokhtenko, E V; Churnosov, M I; Solodilova, M A; Polonikov, A V

    2015-11-01

    Inconsistent results of association studies investigated the role of glutathione S-transferase genes in idiopathic male infertility may be explained by ethnical differences in gene-gene and gene-environment interactions. In this study, we investigated a joint contribution of GSTM1, GSTT1 and GSTP1 gene polymorphisms and cigarette smoking to the risk of idiopathic infertility in Russian men. DNA samples from 203 infertile and 227 fertile men were genotyped by a multiplex polymerase chain reaction (GSTM1 and GSTT1 deletions) and PCR-restriction fragment length polymorphism (GSTP1 I105V) methods. The GSTP1 genotype 105IV was associated with increased risk of male infertility (OR = 1.50 95% CI 1.02-2.20 P = 0.04). Genotype combinations GSTP1 105II/GSTT1 del (G1), GSTM1 del/GSTT1 del (G2) and GSTM1 + /GSTT1 del (G3) were associated with decreased risk of male infertility (P ≤ 0.003), whereas a genotype combination GSTP1 105IV/GSTT1 + (G4) was associated with increased disease risk (P = 0.001). The genotype combinations G3 and G4 showed a significant association with infertility in smokers; however, nonsmokers carriers did show the disease risk. In conclusion, GSTM1, GSTT1 and GSTP1 genes are collectively involved in the development of idiopathic male infertility and their phenotypic effects on the disease risk are potentiated by cigarette smoking.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Cholinesterase and glutathione-S-transferase activities in freshwater invertebrates as biomarkers to assess pesticide contamination.

    PubMed

    Domingues, Inês; Agra, Ana Raquel; Monaghan, Kieran; Soares, Amadeu M V M; Nogueira, António J A

    2010-01-01

    Studies investigating the use of biomarkers in pesticide risk assessment have greatly increased in recent years; however, issues concerning the ecological meaning of enzymatic responses have proved controversial. Ideally a good biomarker response should be modulated by the environmental contaminants alone and demonstrate a predictable behavior towards certain types of toxins. As these premises are rarely observed, the present study aims to outline research that has contributed to an understanding of the behavior of two widely used biomarkers, cholinesterase and glutathione-S-transferase, describing environmental and biotic factors that affect their response in freshwater invertebrates. Studies were performed in the main classes of aquatic invertebrates with these biomarkers and conclusions were reached concerning their behavior towards the main classes of pesticides. Links between biomarker responses and conventional endpoints were evaluated so that ecological relevance could be attributed to enzymatic responses. Toxicity of mixtures was investigated, and cases of synergism and antagonism were pointed out as factors changing the expected toxicity of aquatic systems and leading to misinterpretations of biomarker responses. Finally, the use of biomarkers as a tool for biomonitoring and in situ assays was investigated, with discussion of advantages and disadvantages of their use.

  16. 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.

  17. 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.

  18. 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

  19. Conjugation of 4-hydroxynonenal by largemouth bass (Micropterus salmoides) glutathione S-transferases.

    PubMed

    Pham, Robert T; Gardner, James L; Gallagher, Evan P

    2002-01-01

    The glutathione S-transferases (GST) are a major group of conjugative enzymes involved in the detoxification of electrophilic compounds and products of oxidative stress. We have previously described the kinetics of hepatic GST conjugation in largemouth bass using a variety of synthetic GST reference substrates. In the present study, we investigated the ability of largemouth bass hepatic GSTs to conjugate 4-hydroxynon-2-enal (4HNE), a mutagenic and cytotoxic alpha-beta-unsaturated aldehyde produced during oxidative injury. Hepatic cytosolic fractions from largemouth bass rapidly catalyzed GSH-dependent 4HNE conjugation, with the rate of GST-4HNE conjugation in bass liver exceeding those of several other mammalian and aquatic species. No apparent sex-related differences in GST-4HNE activity were observed among adult bass. SDS-PAGE and Western blotting analysis of GSH affinity-purified bass liver cytosolic GST revealed the presence of two major GST subunits of approximately 30 and 27 KDa that exhibited slight cross-reactivity when probed with a rat alpha class GST antibody, but not to rat mu, pi or theta class GST. The rapid conjugation of 4HNE by hepatic GST suggests an important role for GSTs in protecting against peroxidation of polyunsaturated fatty acids in bass liver.

  20. 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

  1. 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.

  2. Field evaluation of a recombinant glutathione S-transferase-based pyrethroid quantification assay.

    PubMed

    Enayati, Ahmad Ali; Lengeler, Christian; Erlanger, Tobias; Hemingway, Janet

    2005-05-01

    A recombinant glutathione S-transferase (GST)-based pyrethroid quantification assay was field-tested in Ifakara, Tanzania. Initial laboratory tests suggested that all reagents used in the assay should be sufficiently stable for field use, provided that domestic refrigeration facilities were available. Insecticide-impregnated bednets were collected from a region where a social marketing programme was in progress. A total of 100 bednets were collected and the assay plus standard HPLC analysis was performed on the residues extracted from four replicate areas of each net. Insecticide residue estimations for assays performed on white and pale green bednet samples were accurate when compared with residue analysis by HPLC. However, for dark green or blue bednets, there was no correlation between the GST-based assay and HPLC pyrethroid quantification results. The assay failure with the dark coloured nets was caused by the extraction of the dyes along with the insecticide, which subsequently interfered with the GST assay. When the same samples were analysed by HPLC, the dyes were separated from the insecticide by reverse phase column chromatography and hence did not affect the results. PMID:15780344

  3. 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

  4. Characterization and Evolutionary Implications of the Triad Asp-Xxx-Glu in Group II Phosphopantetheinyl Transferases

    PubMed Central

    Wang, Yue-Yue; Li, Yu-Dong; Liu, Jian-Bo; Ran, Xin-Xin; Guo, Yuan-Yang; Ren, Ni-Ni; Chen, Xin; Jiang, Hui; Li, Yong-Quan

    2014-01-01

    Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes. PMID:25036863

  5. 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

  6. 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

  7. Synthesis of isotopically labeled P-site substrates for the ribosomal peptidyl transferase reaction

    PubMed Central

    Zhong, Minghong

    2010-01-01

    Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb,1 have been designed and synthesized in 26–350020steps. These include individual isotopic substitution at the α-proton, carbonyl carbon, and carbonyl oxygen of the amino acid, the O2' and O3' of the adenosine, and a remote label in the N3 and N4 of both cytidines. These isotopomers were synthesized by coupling cytidylyl-(3'5')-cytidine phosphoramidite isotopomers, as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic enrichment is higher than 99% for 1-13C (Phe), 2-2H (Phe), and 3,4-15N2 (cytidine), 93% for 2'/3'- 18O (adenosine), and 64% for 1-18O (Phe). A new synthesis of highly enriched [1-18O2] phenylalanine has been developed. The synthesis of [3'-18O] adenosine was improved by Lewis acid aided regioselective ring opening of the epoxide and by an economical SN2-SN2 method with high isotopic enrichment (93%). Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes, including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes. PMID:18081346

  8. Isolation and characterization of two mouse Pi-class glutathione S-transferase genes.

    PubMed Central

    Bammler, T K; Smith, C A; Wolf, C R

    1994-01-01

    Pi-class glutathione S-transferases (GSTs) play an important role in the detoxification of chemical toxins and mutagens and are implicated in neoplastic development and drug resistance. In all species characterized to date, only one functional Pi-class GST gene has been described. In this report we have identified two actively transcribed murine Pi-class GST genes, Gst p-1 and Gst p-2. The coding regions of Gst p-1 and the mouse Pi-class GST cDNA (GST-II) reported by Hatayama, Satoh and Satoh (1990) (Nucleic Acids Res. 18, 4606) are identical, whereas Gst p-2 encodes a protein that has not been described previously. The two genes are approximately 3 kb long and contain seven exons interrupted by six introns. In addition to a TATA box and a sequence motif matching the phorbol-ester-responsive element, the promoters of Gst p-1 and Gst p-2 exhibit one and two G+C boxes (GGGCGG) respectively. The cDNAs of the two genes were isolated from total liver RNA using reverse PCR. The peptide sequence deduced from the cDNAs share 97% identity and differ in six amino acids. Both genes are transcribed at significantly higher levels in male mouse liver than in female, and Gst p-1 mRNA is more abundant in both sexes than Gst p-2. Images Figure 4 Figure 5 PMID:8135745

  9. 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

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

    PubMed Central

    Hatef, Azadeh; Yufa, Roman

    2015-01-01

    Abstract 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

  11. Effect of cyclodextrin glycosyl transferase [corrected] on dough rheology and bread quality from rice flour.

    PubMed

    Gujral, Hardeep Singh; Guardiola, Ignacio; Carbonell, José Vicente; Rosell, Cristina M

    2003-06-18

    Gluten-free breads are usually characterized by deficient quality characteristics as compared to wheat breads. Problems related to volume and crumb texture are associated with gluten-free breads even when rice flour is used, which seems to be the best raw material for this type of bread. The potential use of cyclodextrin glycosyl transferase (CGTase) as a rice bread improver is presented. The effect of CGTase addition to rice flour on dough rheology and bread quality was investigated. In addition, an experimental design was developed to optimize the levels of CGTase, hydroxypropylmethylcellulose (HPMC), and oil. The addition of CGTase produced a reduction in the dough consistency and also in the elastic modulus. With regard to the rice bread quality, better specific volume, shape index, and crumb texture were obtained. The amount of cyclodextrins in the bread crumb was quantified to explain the action of this enzyme. The data indicate that the improving effect of the CGTase results from a combination of its hydrolyzing and cyclizing activities, the latter being responsible for the release of cyclodextrins, which have the ability to form complexes with lipids and proteins.

  12. Role of Carnitine Acetyl Transferase in Regulation of Nitric Oxide Signaling in Pulmonary Arterial Endothelial Cells

    PubMed Central

    Sharma, Shruti; Sun, Xutong; Agarwal, Saurabh; Rafikov, Ruslan; Dasarathy, Sridevi; Kumar, Sanjiv; Black, Stephen M.

    2013-01-01

    Congenital heart defects with increased pulmonary blood flow (PBF) result in pulmonary endothelial dysfunction that is dependent, at least in part, on decreases in nitric oxide (NO) signaling. Utilizing a lamb model with left-to-right shunting of blood and increased PBF that mimics the human disease, we have recently shown that a disruption in carnitine homeostasis, due to a decreased carnitine acetyl transferase (CrAT) activity, correlates with decreased bioavailable NO. Thus, we undertook this study to test the hypothesis that the CrAT enzyme plays a major role in regulating NO signaling through its effect on mitochondrial function. We utilized the siRNA gene knockdown approach to mimic the effect of decreased CrAT activity in pulmonary arterial endothelial cells (PAEC). Our data indicate that silencing the CrAT gene disrupted cellular carnitine homeostasis, reduced the expression of mitochondrial superoxide dismutase-and resulted in an increase in oxidative stress within the mitochondrion. CrAT gene silencing also disrupted mitochondrial bioenergetics resulting in reduced ATP generation and decreased NO signaling secondary to a reduction in eNOS/Hsp90 interactions. Thus, this study links the disruption of carnitine homeostasis to the loss of NO signaling observed in children with CHD. Preserving carnitine homeostasis may have important clinical implications that warrant further investigation. PMID:23344032

  13. 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

  14. 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

  15. Protein–Polymer Conjugation via Ligand Affinity and Photoactivation of Glutathione S-Transferase

    PubMed Central

    2014-01-01

    A photoactivated, site-selective conjugation of poly(ethylene glycol) (PEG) to the glutathione (GSH) binding pocket of glutathione S-transferase (GST) is described. To achieve this, a GSH analogue (GSH-BP) was designed and chemically synthesized with three functionalities: (1) the binding affinity of GSH to GST, (2) a free thiol for polymer functionalization, and (3) a photoreactive benzophenone (BP) component. Different molecular weights (2 kDa, 5 kDa, and 20 kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed conjugation efficiencies between 52% and 76% to GST. Diazirine (DA) PEG were also prepared but gave conjugation yields lower than for GSBP-PEGs. PEGs with different end-groups were also synthesized to validate the importance of each component in the end-group design. End-groups included glutathione (GS-PEG) and benzophenone (BP-PEG). Results showed that both GSH and BP were crucial for successful conjugation to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins were investigated, including bovine serum albumin (BSA), lysozyme (Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure specific binding to GST. By combining noncovalent and covalent interactions, we have developed a new phototriggered protein–polymer conjugation method that is generally applicable to GST-fusion proteins. PMID:25315970

  16. 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

  17. 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.

  18. Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass.

    PubMed Central

    Foley, V; Sheehan, D

    1998-01-01

    Two similar glutathione S-transferases (GSTs), which do not bind to glutathione- or S-hexylglutathione-agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243-248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme. PMID:9677348

  19. A study of gender, strain and age differences in mouse liver glutathione-S-transferase.

    PubMed

    Egaas, E; Falls, J G; Dauterman, W C

    1995-01-01

    The hepatic cytosolic glutathione S-transferase (GST) activity in four strains of the mouse and one strain of the rat was studied with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethachrynic acid (ETHA), cumene hydroperoxide (CU) and atrazine as the in vitro substrates. In the mouse, significant gender, strain and age-related differences in the GST activity towards CDNB and atrazine were found between adolescent and sexually mature males and females of the CD-1, C57BL/6, DBA/2 and Swiss-Webster strains, and the differences were larger with atrazine as the substrate. With DCNB and CU a similar tendency was observed, however not significant for all strains. The GST activity towards ETHA was also gender and strain specific, but revealed no age-related differences. The herbicide atrazine seems to be a useful substrate in the study of strain and age-related differences in the mouse GST class Pi.

  20. Anthranilate phosphoribosyl transferase (TrpD) generates phosphoribosylamine for thiamine synthesis from enamines and phosphoribosyl pyrophosphate.

    PubMed

    Lambrecht, Jennifer A; Downs, Diana M

    2013-01-18

    Anthranilate phosphoribosyl transferase (TrpD) has been well characterized for its role in the tryptophan biosynthetic pathway. Here, we characterized a new reaction catalyzed by TrpD that resulted in the formation of the purine/thiamine intermediate metabolite phosphoribosylamine (PRA). The data showed that 4- and 5-carbon enamines served as substrates for TrpD, and the reaction product was predicted to be a phosphoribosyl-enamine adduct. Isotopic labeling data indicated that the TrpD reaction product was hydrolyzed to PRA. Variants of TrpD that were proficient for tryptophan synthesis were unable to support PRA formation in vivo in Salmonella enterica. These protein variants had substitutions at residues that contributed to binding substrates anthranilate or phosphoribosyl pyrophosphate (PRPP). Taken together the data herein identified a new reaction catalyzed by a well-characterized biosynthetic enzyme, and both illustrated the robustness of the metabolic network and identified a role for an enamine that accumulates in the absence of reactive intermediate deaminase RidA.

  1. 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

  2. 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.

  3. Increased cytogenetic damage in smokers deficient in glutathione S-transferase isozyme mu.

    PubMed

    van Poppel, G; de Vogel, N; van Balderen, P J; Kok, F J

    1992-02-01

    Reduced expression of the mu-isozyme of glutathione S-transferase (GST; EC 2.5.1.18) has been associated with increased lung cancer risk. We studied the association between GST-mu expression and DNA damage as measured by sister chromatid exchanges (SCE) in healthy male smokers. SCE levels were higher in the 71 GST-mu-deficient smokers compared to the 83 non-deficient smokers (5.24 versus 4.97 SCE/lymphocyte; P = 0.09). In smokers having high plasma cotinine levels (greater than median of 315 ng/ml), this mu-related difference was more pronounced (5.50 versus 4.97; P = 0.01), whereas it was absent in smokers having low cotinine levels (4.95 versus 4.97; P = 0.92). Increased cytogenetic damage in GST-mu-deficient heavy smokers may thus explain the association between GST-mu expression and lung cancer. PMID:1740022

  4. 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

  5. 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

  6. Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay.

    PubMed

    Ganiere-Monteil, C; Pineau, A; Kergueris, M F; Azoulay, C; Bourin, M

    1999-04-30

    A new liquid-liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH4Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5-250 ng ml(-1) (r> or =0.997, slope=1.497, intercept=-0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml(-1), respectively. The coefficients of variation were < or =6.1% for within-day assay (n=6) and < or =9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (7 =70). The results ranged from 7.8 to 27.8 nmol h(-1) ml(-1) packed red blood cells and the frequency distribution histogram is similar to that previously published.

  7. 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

  8. 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

  9. Mechanistic evaluation and transcriptional signature of a glutathione S-transferase omega 1 inhibitor

    PubMed Central

    Ramkumar, Kavya; Samanta, Soma; Kyani, Anahita; Yang, Suhui; Tamura, Shuzo; Ziemke, Elizabeth; Stuckey, Jeanne A.; Li, Si; Chinnaswamy, Krishnapriya; Otake, Hiroyuki; Debnath, Bikash; Yarovenko, Vladimir; Sebolt-Leopold, Judith S.; Ljungman, Mats; Neamati, Nouri

    2016-01-01

    Glutathione S-transferase omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we show that silencing of GSTO1 with siRNA significantly impairs cancer cell viability, validating GSTO1 as a potential new target in oncology. We report on the development and characterization of a series of chloroacetamide-containing potent GSTO1 inhibitors. Co-crystal structures of GSTO1 with our inhibitors demonstrate covalent binding to the active site cysteine. These potent GSTO1 inhibitors suppress cancer cell growth, enhance the cytotoxic effects of cisplatin and inhibit tumour growth in colon cancer models as single agent. Bru-seq-based transcription profiling unravelled novel roles for GSTO1 in cholesterol metabolism, oxidative and endoplasmic stress responses, cytoskeleton and cell migration. Our findings demonstrate the therapeutic utility of GSTO1 inhibitors as anticancer agents and identify the novel cellular pathways under GSTO1 regulation in colorectal cancer. PMID:27703239

  10. Human Polymorphisms in the Glutathione Transferase Zeta 1/Maleylacetoacetate Isomerase Gene Influence the Toxicokinetics of Dichloroacetate

    PubMed Central

    Shroads, Albert L.; Langaee, Taimour; Coats, Bonnie S.; Kurtz, Tracie L.; Bullock, John R.; Weithorn, David; Gong, Yan; Wagner, David A.; Ostrov, David A.; Johnson, Julie A.; Stacpoole, Peter W.

    2013-01-01

    Dichloroacetate (DCA), a chemical relevant to environmental science and allopathic medicine, is dehalogenated by the bifunctional enzyme glutathione transferase zeta (GSTz1) maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway. The authors postulated that polymorphisms in GSTz1/MAAI modify the toxicokinetics of DCA. GSTz1/MAAI haplotype significantly affected the kinetics and biotransformation of 1,2-13C-DCA when it was administered at either environmentally (μg/kg/d) or clinically (mg/kg/d) relevant doses. GSTz1/MAAI haplotype also influenced the urinary accumulation of potentially toxic tyrosine metabolites. Atomic modeling revealed that GSTz1/MAAI variants associated with the slowest rates of DCA metabolism induced structural changes in the enzyme homodimer, predicting protein instability or abnormal protein-protein interactions. Knowledge of the GSTz1/MAAI haplotype can be used prospectively to identify individuals at potential risk of DCA’s adverse side effects from environmental or clinical exposure or who may exhibit aberrant amino acid metabolism in response to dietary protein. PMID:21642471

  11. 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

  12. Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells.

    PubMed

    Loraine, A E; Yalovsky, S; Fabry, S; Gruissem, W

    1996-04-01

    Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport. To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase). We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells. The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence. LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase. LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato. LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit. The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening.

  13. O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat

    PubMed Central

    Ruan, Hai-Bin; Dietrich, Marcelo O.; Liu, Zhong-Wu; Zimmer, Marcelo R.; Li, Min-Dian; Singh, Jay Prakash; Zhang, Kaisi; Yin, Ruonan; Wu, Jing; Horvath, Tamas L.; Yang, Xiaoyong

    2014-01-01

    SUMMARY Induction of beige cells causes the browning of white fat and improves energy metabolism. However, the central mechanism that controls adipose tissue browning and its physiological relevance are largely unknown. Here we demonstrate that fasting and chemical-genetic activation of orexigenic AgRP neurons in the hypothalamus suppress the browning of white fat. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates fundamental cellular processes. The levels of O-GlcNAc transferase (OGT) and O-GlcNAc modification are enriched in AgRP neurons and are elevated by fasting. Genetic ablation of OGT in AgRP neurons inhibits neuronal excitability through the voltage-dependent potassium channel, promotes white adipose tissue browning, and protects mice against diet-induced obesity and insulin resistance. These data reveal adipose tissue browning as a highly dynamic physiological process under central control, in which O-GlcNAc signaling in AgRP neurons is essential for suppressing thermogenesis to conserve energy in response to fasting. PMID:25303527

  14. Polyprenyl lipid synthesis in mammalian cells expressing human cis-prenyl transferase.

    PubMed

    Jones, Jullian; Viswanathan, Karthik; Krag, Sharon S; Betenbaugh, Michael J

    2005-06-01

    The level of cis-prenyl transferase activity has been implicated in controlling the level of biosynthesis of dolichol and dolichol intermediates. In this study, we isolated a cDNA encoding a human CPT (GenBank Accession No. ), which had substantial homology to other CPT isolated from human brain, bacteria, Arabidopsis, and Saccharomyces cerevisiae. Expression of this cDNA in two different insect cell lines confirmed the functionality of the protein in an in vitro assay. Western blot analysis revealed an expressed protein of approximately 38 kDa in HEK293 cells. Overexpression of the protein in HEK293 cells resulted in an increase in the level of total prenol in vivo. Furthermore, product characterization by thin layer chromatography (TLC) confirmed that the major product was a long-chain prenol with a chain length of 95 carbons. These results suggest a regulatory relationship between CPT activity and dolichol biosynthesis, and may implicate CPT in the levels of dolichol-oligosaccharide intermediate biosynthesis.

  15. O-GlcNAc transferase and O-GlcNAcase: achieving target substrate specificity

    PubMed Central

    Nagel, Alexis K.

    2015-01-01

    O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) catalyze the dynamic cycling of intracellular, post-translational O-GlcNAc modification on thousands of Ser/Thr residues of cytosolic, nuclear, and mitochondrial signaling proteins. The identification of O-GlcNAc modified substrates has revealed a functionally diverse set of proteins, and the extent of O-GlcNAcylation fluctuates in response to nutrients and cellular stress. As a result, OGT and OGA are implicated in widespread, nutrient-responsive regulation of numerous signaling pathways and transcriptional programs. These enzymes are required for normal embryonic development and are dysregulated in metabolic and age-related disease states. While a recent surge of interest in the field has contributed to understanding the functional impacts of protein O-GlcNAcylation, little is known about the upstream mechanisms which modulate OGT and OGA substrate targeting. This review focuses on elements of enzyme structure among splice variants, post-translational modification, localization, and regulatory protein interactions which drive the specificity of OGT and OGA toward different subsets of the cellular proteome. Ongoing efforts in this rapidly advancing field are aimed at revealing mechanisms of OGT and OGA regulation to harness the potential therapeutic benefit of manipulating these enzymes’ activities. PMID:25173736

  16. Limonin Methoxylation Influences Induction of Glutathione S-Transferase and Quinone Reductase

    PubMed Central

    PEREZ, JOSE LUIS; JAYAPRAKASHA, G. K.; VALDIVIA, VIOLETA; MUNOZ, DIANA; DANDEKAR, DEEPAK V.; AHMAD, HASSAN; PATIL, BHIMANAGOUDA S.

    2009-01-01

    Previous studies have indicated the chemoprevention potential of citrus limonoids due to the induction of phase II detoxifying enzymes. In the present study, three citrus limonoids were purified and identified from sour orange seeds as limonin, limonin glucoside (LG), deacetylnomilinic acid glucoside (DNAG). In addition, limonin was modified to defuran limonin and limonin 7-methoxime. The structures of these compounds were confirmed by NMR studies. These five compounds were used to investigate the influence of Phase II enzymes in female A/J mice. Our results indicated that the highest induction of Glutathione S-Transferase (GST) activity against 1-chloro-2, 4-dinitrobenzene (CDNB) by DNAG (67%) in lung homogenates followed by limonin-7-methoxime (32%) in treated liver homogenates. Interestingly, the limonin-7-methoxime showed the highest GST activity (270%) in liver against 4-nitroquinoline 1-oxide (4NQO), while the same compound in stomach induced GST by 51% compared to the control. DNAG treated group induced 55% in stomach homogenates. Another Phase II enzyme, quinone reductase (QR), was significantly induced by limonin-7-methoxime by 65 and 32% in liver and lung homogenates, respectively. Defuran limonin, induced QR in lung homogenates by 45%. Our results indicated that modification of the limonin have differential induction of phase II enzymes. These findings are indicative of a possible mechanism for the prevention of cancer by aiding in detoxification of xenobiotics. PMID:19480426

  17. 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.

  18. 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

  19. Characterization and evolutionary implications of the triad Asp-Xxx-Glu in group II phosphopantetheinyl transferases.

    PubMed

    Wang, Yue-Yue; Li, Yu-Dong; Liu, Jian-Bo; Ran, Xin-Xin; Guo, Yuan-Yang; Ren, Ni-Ni; Chen, Xin; Jiang, Hui; Li, Yong-Quan

    2014-01-01

    Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.

  20. Molecular characterization of an anthocyanin-related glutathione S-transferase gene in cyclamen.

    PubMed

    Kitamura, Satoshi; Akita, Yusuke; Ishizaka, Hiroshi; Narumi, Issay; Tanaka, Atsushi

    2012-04-15

    Anthocyanins are a subclass of flavonoids and are a major contributor to flower colors ranging from red to blue and purple. Previous studies in model and ornamental plants indicate a member of the glutathione S-transferase (GST) gene family is involved in vacuolar accumulation of anthocyanins. In order to identify the anthocyanin-related GST in cyclamen, degenerate PCR was performed using total RNA from immature young petals. Four candidates of GSTs (CkmGST1 to CkmGST4) were isolated. Phylogenetic analysis indicated that CkmGST3 was closely related to PhAN9, an anthocyanin-related GST of petunia, and this clade was clustered with other known anthocyanin-related GSTs. Expression analysis at different developmental stages of petals revealed that CkmGST3 was strongly expressed in paler pigmented petals than in fully pigmented petals, in contrast to the constitutive expression of the other three candidates during petal development. This expression pattern of CkmGST3 was correlated with those of other anthocyanin biosynthetic genes such as CkmF3'5'H and CkmDFR2. Molecular complementation of Arabidopsis tt19, a knockout mutant of an anthocyanin-related GST gene, demonstrated that CkmGST3 could complement the anthocyanin-less phenotype of tt19. Transgenic plants that expressed the other three CkmGSTs did not show anthocyanin accumulation. These results indicate CkmGST3 functions in anthocyanin accumulation in cyclamen.

  1. Glutathione S-transferase M1 and P1 metabolic polymorphism and lung cancer predisposition.

    PubMed

    Reszka, E; Wasowicz, W; Rydzynski, K; Szeszenia-Dabrowska, N; Szymczak, W

    2003-01-01

    Individual susceptibility to different environmental agents is expected to be associated with alterations in metabolism of xenobiotics. Thus, genetic polymorphism of glutathione S-transferase (GST) can be recognized as a potential risk modifier in lung cancer development. The distribution of GSTM1 and GSTP1 genotypes was studied in a group of 138 diagnosed lung cancer patients and in 165 controls living in central Poland and RFLP-PCR technique was applied. The frequency of GSTM1 null genotype and GSTP1 Val single and duplicated alleles was similar among patients and controls. GSTM1 homozygous deletion was most prevalent in small-cell carcinoma groups (adjusted odds ratio (OR): 2.32, 95% confidence interval (CI): 0.98-5.52). In patients and controls, GSTM1A genotype was most frequent (34.1% vs. 37.0%). The estimated lung cancer risk for GSTM1 null, GSTP1 Ile/Val and GSTP1 Val/Val combined genotype was 1.44 (95% CI: 0.73-2.83), suggesting the absence of modifying effect of defective GSTM1 and GSTP1 alleles on lung cancer predisposition. PMID:14628089

  2. Computational evidence for the detoxifying mechanism of epsilon class glutathione transferase toward the insecticide DDT.

    PubMed

    Li, Yanwei; Shi, Xiangli; Zhang, Qingzhu; Hu, Jingtian; Chen, Jianmin; Wang, Wenxing

    2014-05-01

    A combined quantum mechanics/molecular mechanics (QM/MM) computation of the detoxifying mechanism of an epsilon class glutathione transferases (GSTs) toward organochlorine insecticide DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, has been carried out. The exponential average barrier of the proton transfer mechanism is 15.2 kcal/mol, which is 27.6 kcal/mol lower than that of the GS-DDT conjugant mechanism. It suggests that the detoxifying reaction proceeds via a proton transfer mechanism where GSH acts as a cofactor rather than a conjugate. The study reveals that the protein environment has a strong effect on the reaction barrier. The experimentally proposed residues Arg112, Glu116 and Phe120 were found to have a strong influence on the detoxifying reaction. The influence of residues Pro13, Cys15, His53, Ile55, Glu67, Ser68, Phe115, and Leu119 was detected as well. It is worth noticing that Ile55 facilitates the detoxifying reaction most. On the basis of the structure of DDT, structure 2, (BrC6H4)2CHCCl3, is the best candidate among all the tested structures in resisting the detoxification of enzyme agGSTe2.

  3. Structural insights into the dehydroascorbate reductase activity of human omega-class glutathione transferases.

    PubMed

    Zhou, Huina; Brock, Joseph; Liu, Dan; Board, Philip G; Oakley, Aaron J

    2012-07-13

    The reduction of dehydroascorbate (DHA) to ascorbic acid (AA) is a vital cellular function. The omega-class glutathione transferases (GSTs) catalyze several reductive reactions in cellular biochemistry, including DHA reduction. In humans, two isozymes (GSTO1-1 and GSTO2-2) with significant DHA reductase (DHAR) activity are found, sharing 64% sequence identity. While the activity of GSTO2-2 is higher, it is significantly more unstable in vitro. We report the first crystal structures of human GSTO2-2, stabilized through site-directed mutagenesis and determined at 1.9 Å resolution in the presence and absence of glutathione (GSH). The structure of a human GSTO1-1 has been determined at 1.7 Å resolution in complex with the reaction product AA, which unexpectedly binds in the G-site, where the glutamyl moiety of GSH binds. The structure suggests a similar mode of ascorbate binding in GSTO2-2. This is the first time that a non-GSH-based reaction product has been observed in the G-site of any GST. AA stacks against a conserved aromatic residue, F34 (equivalent to Y34 in GSTO2-2). Mutation of Y34 to alanine in GSTO2-2 eliminates DHAR activity. From these structures and other biochemical data, we propose a mechanism of substrate binding and catalysis of DHAR activity.

  4. Crystallization and X-ray diffraction studies of glutathione S-transferase from Escherichia coli

    NASA Astrophysics Data System (ADS)

    Nishida, Motohiko; Harada, Shigeharu; Satow, Yoshinori; Inoue, Hideshi; Takahashi, Kenji

    1996-10-01

    Crystals of glutathione S-transferase from Escherichia coli have been obtained by use of polyethylene glycol 6000 as a precipitant. The crystallization was performed in the presence of a glutathione sulfonate inhibitor under the acidic condition, with combination of the sitting-drop vapour-diffusion and the macro-seeding procedures. The crystals are of a thin-plate shape with typical sizes of 1.0 × 0.5 × 0.1 mm, and are stable against X-ray irradiation. They belong to the space group P2 12 12 1 with cell parameters of a = 90.47 Å, b = 93.87 Å and c = 51.10 Å, and diffract X-rays at least up to 2.3 Å resolution. The solvent content is 48% in volume, when a homodimeric molecule of the enzyme is assumed to occupy an asymmetric unit of the crystal. The crystals are suitable for three-dimensional structural studies. Diffraction data of the native crystal have been collected.

  5. 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.

  6. Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase.

    PubMed

    Van Hellemond, J J; Opperdoes, F R; Tielens, A G

    1998-03-17

    Hydrogenosome-containing anaerobic protists, such as the trichomonads, produce large amounts of acetate by an acetate:succinate CoA transferase (ASCT)/succinyl CoA synthetase cycle. The notion that mitochondria and hydrogenosomes may have originated from the same alpha-proteobacterial endosymbiont has led us to look for the presence of a similar metabolic pathway in trypanosomatids because these are the earliest-branching mitochondriate eukaryotes and because they also are known to produce acetate. The mechanism of acetate production in these organisms, however, has remained unknown. Four different members of the trypanosomatid family: promastigotes of Leishmania mexicana mexicana, L. infantum and Phytomonas sp., and procyclics of Trypanosoma brucei were analyzed as well as the parasitic helminth Fasciola hepatica. They all use a mitochondrial ASCT for the production of acetate from acetyl CoA. The succinyl CoA that is produced during acetate formation by ASCT is recycled presumably to succinate by a mitochondrial succinyl CoA synthetase, concomitantly producing ATP from ADP. The ASCT of L. mexicana mexicana promastigotes was further characterized after partial purification of the enzyme. It has a high affinity for acetyl CoA (Km 0.26 mM) and a low affinity for succinate (Km 6.9 mM), which shows that significant acetate production can occur only when high mitochondrial succinate concentrations prevail. This study identifies a metabolic pathway common to mitochondria and hydrogenosomes, which strongly supports a common origin for these two organelles.

  7. Glutathione transferase isoenzymes from Bufo bufo embryos at an early developmental stage.

    PubMed Central

    Di Ilio, C; Aceto, A; Bucciarelli, T; Dragani, B; Angelucci, S; Miranda, M; Poma, A; Amicarelli, F; Barra, D; Federici, G

    1992-01-01

    Six forms of glutathione transferase (GST) were resolved from the cytosolic fraction of Bufo bufo embryos at developmental stage 4 by GSH-Sepharose affinity chromatography followed by f.p.l.c. chromatofocusing in the 9-6 pH range. They have apparent isoelectric points at pH 8.37 (GST I), 8.22 (GST II), 8.10 (GST III), 7.84 (GST IV), 7.37 (GST V) and 7.12 (GST VI), and each displayed an apparent subunit molecular mass of 23 kDa by SDS/PAGE. The Bufo bufo embryo enzymes showed very similar structural, catalytic and immunological properties, as indicated by their substrate-specificities, inhibition characteristics, c.d. spectra, h.p.l.c. elution profiles and immunological reactivities, as well as by their N-terminal amino acid sequences. Although Bufo bufo embryo GSTs do not correspond to any other known GSTs, the results of our experiments indicate that amphibian GSTs could be included in the Pi family of GSTs. This conclusion is supported by the analysis of c.d. spectra, and by the fact that mammalian Pi class GSTs and amphibian GSTs showed about 80% identity in their N-terminal amino acid sequences. Furthermore, antisera prepared against Bufo bufo GST III cross-reacted in immunoblotting analysis with Pi class GSTs, and vice versa. Images Fig. 2. Fig. 3. PMID:1567369

  8. 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

  9. Glutathione s-transferase M1 and T1 genetic polymorphisms in Iranian patients with glaucoma

    PubMed Central

    Safa, Fatemeh Kazemi; Shahsavari, Gholamreza; Abyaneh, Reza Zare

    2014-01-01

    Objective(s): Glaucoma is the second leading cause of blindness and it is related to oxidative stress based on numerous studies. Glutathione S-transferases (GSTs) are members of multigenic family, which have important role in cells as an antioxidant. In the present study, we examined the polymorphism of GSTT1 and GSTM1 deletion genotypes (T0M1, T1M0, and T0M0) in 100 Glaucoma patients (41with primary open angle glaucoma (PCAG), and 59 with primary closed angle glaucoma (POAG)) compared to 100 healthy subjects. Materials and Methods: GSTM1and GSTT1 polymorphisms were determined by multiplex polymerase chain reaction. Results: GSTM1 and GSTT1 null deletions genotypes were determined in 22 (53.7%) and 7 (17.1%) patients with PCAG and 34 (34%) and 15 (15%) in healthy subjects. Comparison between patients and healthy subjects regarding GSTM1 and GSTT1 genotypes revealed increase of GSTM1 null deletions genotypes in patients with PCAG (P=0.03). Conclusion: It was concluded that the increased frequencies of GSTM1 null in patients with PCAG could be a risk factor for incidence of PCAG in the Iranian population. PMID:24967061

  10. 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

  11. Antenna-specific glutathione S-transferase in male silkmoth Bombyx mori.

    PubMed

    Tan, Xiang; Hu, Xiao-Ming; Zhong, Xiao-Wu; Chen, Quan-Mei; Xia, Qing-You; Zhao, Ping

    2014-04-29

    Glutathione S-transferases (GSTs) are multifunctional enzymes that are widely distributed in different species. GSTs detoxify exogenous and endogenous substances by conjugation to reduced glutathione. We characterized BmGSTD4, an antenna-specific GST, in male silkmoths. The full-length mRNA of Bmgstd4 was cloned by RACE-PCR and contained an open reading frame of 738 bp encoding a 245 amino acid protein. The antenna specificity of BmGSTD4 was validated at the mRNA and protein levels and BmGSTD4 was shown to localize in the sensillum of male silkmoth antennae. Homology modeling and multi-sequence alignment suggested that BmGSTD4 was a typical GST belonging to the δ class and had a canonical GST fold with a conserved N-terminus, including a glutathione-binding site and a C-terminal domain harboring a hydrophobic substrate-binding site. Restricted expression of BmGSTD4 in silkmoth antennae combined with GST activity suggested that BmGSTD4 was involved in the detoxification of harmful chemicals.

  12. 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.

  13. 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

  14. Inhibitory mechanism of novel inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae.

    PubMed

    Jin, Bong-Suk; Han, Seong-Gu; Lee, Won-Kyu; Ryoo, Sung Weon; Lee, Sang Jae; Suh, Se-Won; Yu, Yeon Gyu

    2009-12-01

    Bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolphyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first step of bacterial cell wall synthesis. We identified thimerosal, thiram, and ebselen as effective inhibitors of Heamophilus influenzae MurA by screening a chemical library that consisted of a wide range of bioactive compounds. When MurA was preincubated with these inhibitors, their 50% inhibitory concentrations (IC50s) were found to range from 0.1 to 0.7 microM. In particular, thimerosal suppressed the growth of several different Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium at a concentration range of 1-2 microg/ml. These inhibitors covalently modified the cysteine residue near the active site of MurA. This modification changed the open conformation of MurA to a more closed configuration, which may have prevented the necessary conformational change from occurring during the enzyme reaction.

  15. Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: focus on a new glutathione transferase.

    PubMed

    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; Morel-Rouhier, Mélanie

    2014-10-01

    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.

  16. Glutathione S-transferase localization in aflatoxin B1-treated rat livers.

    PubMed

    Harrison, D J; May, L; Hayes, J D; Neal, G E

    1990-06-01

    Overexpression of detoxication enzymes is associated with the development of drug-resistant, preneoplastic nodules in the carcinogen-treated rat liver. The most consistent marker of preneoplasia in many experimental models is increased expression of the pi-class glutathione S-transferase (GST) YfYf. We have confirmed by immunostaining that the pi-class GST is overexpressed in aflatoxin B1-induced preneoplastic nodules and liver tumours in rats. However, pi-class GST YfYf has low activity against aflatoxin B1-8,9-epoxide, and most activity against this cytotoxic and genotoxic metabolite is associated with the alpha-class GSTs YaYa, YaYc and YcYc. We have demonstrated that there is also a consistent increase in the alpha-class GSTs in this model. It seems likely that the overexpression of the Ya and Yc subunits, rather than increased levels of the pi-class GST YfYf, is responsible for the acquisition of a drug-resistant phenotype in rat liver preneoplastic nodules and tumours induced by aflatoxin B1. PMID:2112061

  17. 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

  18. Transcriptional Responses of Glutathione Transferase Genes in Ruditapes philippinarum Exposed to Microcystin-LR

    PubMed Central

    Reis, Bruno; Carneiro, Mariana; Machado, João; Azevedo, Joana; Vasconcelos, Vitor; Martins, José Carlos

    2015-01-01

    Glutathione Transferases (GSTs) are phase II detoxification enzymes known to be involved in the molecular response against microcystins (MCs) induced toxicity. However, the individual role of the several GST isoforms in the MC detoxification process is still unknown. In this study, the time-dependent changes on gene expression of several GST isoforms (pi, mu, sigma 1, sigma 2) in parallel with enzymatic activity of total GST were investigated in gills and hepatopancreas of the bivalve Ruditapes philippinarum exposed to pure MC-LR (10 and 100 µg/L). No significant changes in GST enzyme activities were found on both organs. In contrast, MC-LR affected the transcriptional activities of these detoxification enzymes both in gills and hepatopancreas. GST transcriptional changes in gills promoted by MC-LR were characterized by an early (12 h) induction of mu and sigma 1 transcripts. On the other hand, the GST transcriptional changes in hepatopancreas were characterized by a later induction (48 h) of mu transcript, but also by an early inhibition (6 h) of the four transcripts. The different transcription patterns obtained for the tested GST isoforms in this study highlight the potential divergent physiological roles played by these isoenzymes during the detoxification of MC-LR. PMID:25884330

  19. 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

  20. 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.

  1. Characterization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.).

    PubMed

    James, D W; Jones, R L

    1979-12-01

    The peripheral root cap cells of corn (cv. SX-17A) secrete a fucose-rich, high molecular weight, polysaccharide slime via the dictyosome pathway. To study the synthesis of this polysaccharide, a technique for isolating and assaying GDP-fucose:polysaccharide fucosyl transferase activity was developed. Corn roots were excised from germinated seeds, incubated 12 hours at 10 C in water, and ground in 100 millimolar Tris or Pipes buffer (pH 7.0) with or without 0.5 molar sucrose. The membrane-bound enzyme was solubilized by sonication in the presence of 2 molar urea and 1.5% (v/v) Triton X-100 and assayed by monitoring the incorporation of GDP-[(14)C]fucose into endogenous acceptors. Optimum enzyme activity is expressed at pH 7.0 and 30 C in the presence of 0.8% (v/v) Triton X-100. The enzyme does not require divalent cations for activation and is inhibited by concentrations of MnCl(2) or MgCl(2) greater than 1 millimolar. Corn root cap slime will serve as an exogenous acceptor for the enzyme if it is first hydrolyzed in 5 millimolar trifluoroacetic acid for 60 minutes at 18 pounds per square inch, 121 C. This procedure prepares the acceptor by removing terminal fucose residues from the slime molecule. Kinetics of fucose release during hydrolysis of native slime and in vitro synthesized product suggests that the two polymers possess similar linkages to fucose.

  2. Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris.

    PubMed Central

    Marzocca, M P; Harding, N E; Petroni, E A; Cleary, J M; Ielpi, L

    1991-01-01

    Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N.E. Harding, J.M. Cleary, D.K. Cabañas, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tetr) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain. PMID:1657892

  3. Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris.

    PubMed

    Marzocca, M P; Harding, N E; Petroni, E A; Cleary, J M; Ielpi, L

    1991-12-01

    Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N.E. Harding, J.M. Cleary, D.K. Cabañas, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tetr) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain. PMID:1657892

  4. Glutathione S-transferase P1 ILE105Val polymorphism in occupationally exposed bladder cancer cases.

    PubMed

    Kopps, Silke; Angeli-Greaves, Miriam; Blaszkewicz, Meinolf; Prager, Hans-Martin; Roemer, Hermann C; Lohlein, Dietrich; Weistenhofer, Wobbeke; Bolt, Hermann M; Golka, Klaus

    2008-01-01

    The genotype glutathione S-transferase P1 (GSTP1) influences the risk for bladder cancer among Chinese workers occupationally exposed to benzidine. Studies of Caucasian bladder cancer cases without known occupational exposures showed conflicting results. Research was thus conducted to define the role of GSTP1 genotypes in Caucasian bladder cancer cases with an occupational history of exposure to aromatic amines. DNA from 143 cases reported to the Industrial Professional Associations (Berufsgenossenschaften) in Germany from 1996 to 2004, who had contracted urothelial cancer due to occupational exposure, and 196 patients from one Department of Surgery in Dortmund, without known malignancy in their medical history, were genotyped using real-time polymerase chain reaction (PCR) (LightCycler) in relation to GSTP1 A1578G (Ile105Val) polymorphism. Among the subjects with bladder cancer, 46% presented the AA genotype, 39% the AG genotype, and 15% the GG genotype. In the surgical (noncancer) control group analyzed, 42% presented the AA genotype, 42% the AG genotype, and 16% the GG genotype. A subgroup of bladder cancer cases, represented by 46 painters, showed a distribution of 41% of the AA genotype, 48% of the AG genotype, and 11% of the GG genotype. Data indicated that in Caucasians exposed to aromatic amines the GSTP1 A1578G polymorphism did not appear to play a significant role as a predisposing factor for bladder cancer incidence.

  5. Response to adjuvant chemotherapy in primary breast cancer: no correlation with expression of glutathione S-transferases.

    PubMed Central

    Peters, W. H.; Roelofs, H. M.; van Putten, W. L.; Jansen, J. B.; Klijn, J. G.; Foekens, J. A.

    1993-01-01

    Of 139 node-positive breast cancer patients treated with adjuvant chemotherapy, the pre-treatment levels of glutathione S-transferase (GST) classes alpha, mu and pi, were determined by immuno-quantification on Western blots in cytosols of the primary tumours. Their expression was studied with respect to cytosolic oestrogen-receptor, progesterone-receptor and cathepsin D levels, and to the length of disease-free survival. GST class pi was negatively correlated with oestrogen receptor and progesterone receptor, and positively correlated with cathepsin D. There was no correlation between GST isoenzymes and the length of disease-free survival. These data suggest that glutathione S-transferases are not useful as markers to predict the response to adjuvant chemotherapy in human breast cancer. Images Figure 1 PMID:8318426

  6. Action of glycosyl transferases upon "Bombay" (Oh) erythrocytes. Conversion to cells showing blood-group H and A specificities.

    PubMed

    Schenkel-Brunner, H; Prohaska, R; Tuppy, H

    1975-08-15

    Individuals of the rare "Bombay" (Oh) blood-group phenotype lacking, due to a genetic defect, the alpha(1-2)fucosyl transferase, which is responsible for converting blood-group H precursor substances to H-specific structures. Treatment with GDP-fucose and alpha(1-2)fucosyl transferase prepared from gastric mucosa of O individuals to transform native or ficin-treated "Bombay" erythrocytes into cells phenotypically resembling O cells. The transformation was achieved, however, after prior incubation of the "Bombay" erythrocytes with neuraminidase, indicating that blood-group H precursor molecules on the surface of these cells are masked by sialyl residues. Blood-group A specificity was conferred upon neuraminidase-treated "Bombay" cells by enzymatic transfer of alpha-N-acetylgalactosamine residues, in addition to alpha-fucose residues.

  7. 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

  8. Effects of glutathione S-transferase M1 and T1 deletions on epilepsy risk among a Tunisian population.

    PubMed

    Chbili, Chahra; B'chir, Fatma; Ben Fredj, Maha; Saguem, Bochra-Nourhène; Ben Amor, Sana; Ben Ammou, Sofiene; Saguem, Saad

    2014-09-01

    Glutathione-S-transferases enzymes are involved in the detoxification of several endogenous and exogenous substances. In this present study, we evaluated the effects of two glutathione-S-transferase polymorphisms, (GSTM1 and GSTT1) on epilepsy risk susceptibility in a Tunisian population. These polymorphisms were analyzed in 229 healthy subjects and 98 patients with epilepsy, using a polymerase chain reaction (PCR). Odds ratio (ORs) was used for analyzing results. The study results demonstrated that individuals with the GSTM1 null genotype were at an increased risk of developing epilepsy [OR=3.80, 95% confidence interval (CI) (2.15-4.78)], whereas no significant effects were observed between individuals with GSTT1 null genotype and epilepsy risk [OR=1.15, 95% CI (0.62-2.12)]. These genotyping finding revealed that the absence of GSTM1 activity could be contributor factor for the development of epilepsy disease.

  9. 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

  10. Identification and subcellular localization of TcHIP, a putative Golgi zDHHC palmitoyl transferase of Trypanosoma cruzi.

    PubMed

    Batista, Cassiano Martin; Kalb, Ligia Cristina; Moreira, Claudia Maria do Nascimento; Batista, Guilherme Tadashi Hono; Eger, Iriane; Soares, Maurilio José

    2013-05-01

    Protein palmitoylation is a post-translational modification that contributes to determining protein localization and function. Palmitoylation has been described in trypanosomatid protozoa, but no zDHHC palmitoyl transferase has been identified in Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. In this study we identify and show the subcellular localization of TcHIP (Tc00.1047053508199.50), a putative T. cruzi zDHHC palmitoyl transferase. Analysis of the deduced protein sequence indicates that it contains ankyrin repeats (Ank and Ank2) and the zDHHC conserved domain, typical of zDHHC palmitoyl transferases. A TcHIP polyclonal antiserum obtained from mice immunized with the purified recombinant protein was used to study the presence and subcellular localization of the native enzyme. In western blots this antiserum recognized a protein of about 95 kDa, consistent with the predicted molecular mass of TcHIP (95.4 kDa), in whole extracts of T. cruzi epimastigotes, metacyclic trypomastigotes and intracellular amastigotes. Immunolocalization by confocal microscopy showed TcHIP labeling at the Golgi complex, co-localizing with the T. cruzi Golgi marker TcRab7-GFP. Transfectant T. cruzi epimastigotes containing a construct encoding TcHIP fused to proteins A and C (TcHIP/AC) were obtained. In western blotting experiments, the TcHIP polyclonal antiserum recognized both native and TcHIP/AC proteins in extracts of the transfectants. Confocal microscopy showed co-localization of native TcHIP with TcHIP/AC. These findings demonstrate the presence of a putative zDHHC palmitoyl transferase (TcHIP) containing ankyrin and zDHHC domains in different developmental forms of T. cruzi, and its association with the Golgi complex. PMID:23428831

  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. A fluorescent assay amenable to measuring production of beta-D-glucuronides produced from recombinant UDP-glycosyl transferase enzymes.

    PubMed

    Trubetskoy, O V; Shaw, P M

    1999-05-01

    Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.

  13. An Alternate Pathway of Arsenate Resistance in E. coli Mediated by the Glutathione S-Transferase GstB

    PubMed Central

    2015-01-01

    Microbial arsenate resistance is known to be conferred by specialized oxidoreductase enzymes termed arsenate reductases. We carried out a genetic selection on media supplemented with sodium arsenate for multicopy genes that can confer growth to E. coli mutant cells lacking the gene for arsenate reductase (E. coli ΔarsC). We found that overexpression of glutathione S-transferase B (GstB) complemented the ΔarsC allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type E. coli arsenate reductase, arsenate resistance via GstB was not dependent on reducing equivalents provided by glutaredoxins or a catalytic cysteine residue. Instead, two arginine residues, which presumably coordinate the arsenate substrate within the electrophilic binding site of GstB, were found to be critical for transferase activity. We provide biochemical evidence that GstB acts to directly reduce arsenate to arsenite with reduced glutathione (GSH) as the electron donor. Our results reveal a pathway for the detoxification of arsenate in bacteria that hinges on a previously undescribed function of a bacterial glutathione S-transferase. PMID:25517993

  14. An alternate pathway of arsenate resistance in E. coli mediated by the glutathione S-transferase GstB.

    PubMed

    Chrysostomou, Constantine; Quandt, Erik M; Marshall, Nicholas M; Stone, Everett; Georgiou, George

    2015-03-20

    Microbial arsenate resistance is known to be conferred by specialized oxidoreductase enzymes termed arsenate reductases. We carried out a genetic selection on media supplemented with sodium arsenate for multicopy genes that can confer growth to E. coli mutant cells lacking the gene for arsenate reductase (E. coli ΔarsC). We found that overexpression of glutathione S-transferase B (GstB) complemented the ΔarsC allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type E. coli arsenate reductase, arsenate resistance via GstB was not dependent on reducing equivalents provided by glutaredoxins or a catalytic cysteine residue. Instead, two arginine residues, which presumably coordinate the arsenate substrate within the electrophilic binding site of GstB, were found to be critical for transferase activity. We provide biochemical evidence that GstB acts to directly reduce arsenate to arsenite with reduced glutathione (GSH) as the electron donor. Our results reveal a pathway for the detoxification of arsenate in bacteria that hinges on a previously undescribed function of a bacterial glutathione S-transferase.

  15. 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...

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

    PubMed Central

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

    2014-01-01

    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 understand the function of the acyl-group binding pocket in substrate specificity. PMID:24699651

  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. 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. A Novel Method of Production and Biophysical Characterization of the Catalytic Domain of Yeast Oligosaccharyl Transferase

    PubMed Central

    Huang, Chengdong; Mohanty, Smita; Banerjee, Monimoy

    2010-01-01

    Oligosaccharyl transferase (OT) is a multi-subunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, a robust but novel method of production of pure C-terminal domain of Stt3p at 60∼70 mg/L in E. coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well dispersed 2D {1H-15N}-HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The comparative analysis of the fluorescence and NMR data of the mutant and the wild-type C-terminal domain of Stt3p revealed that the replacement of the key residue Asp518, which is located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures. This observation strongly suggests that Asp518, which was previously proposed to primarily function as a catalytic residue, also plays a critical structural role. Moreover, the activity of the protein was confirmed by Saturation Transfer Difference (STD) and NMR titration studies. PMID:20047336

  20. 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

  1. Interchangeable Domains in the Kdo Transferases of Escherichia coli and Haemophilus influenzae

    PubMed Central

    Chung, Hak Suk; Raetz, Christian R. H.

    2010-01-01

    Kdo2-lipid A, a conserved substructure of lipopolysaccharide, plays critical roles in Gram-negative bacterial survival and interaction with host organisms. Inhibition of Kdo biosynthesis in Escherichia coli results in cell death and accumulation of the tetra-acylated precursor lipid IVA. E. coli KdtA (EcKdtA) is a bi-functional enzyme that transfers two Kdo units from two CMP-Kdo molecules to lipid IVA. In contrast, H. influenzae KdtA (HiKdtA) transfers only one Kdo unit. E. coli CMR300, which lacks Kdo-transferase because of a deletion in kdtA, can be rescued to grow in broth at 37 °C if multiple copies of msbA are provided in trans. MsbA, the inner membrane transporter for nascent lipopolysaccharide, prefers hexa-acylated to tetra-acylated lipid A, but with the excess MsbA present in CMR300, lipid IVA is efficiently exported to the outer membrane. CMR300 is hypersensitive to hydrophobic antibiotics and bile salts, and does not grow at 42 °C. Expressing HiKdtA in CMR300 results in the accumulation of Kdo-lipid IVA in place of lipid IVA without suppression of its growth phenotypes at 30 °C. EcKdtA restores intact lipopolysaccharide, together with normal antibiotic resistance, detergent resistance and growth at 42 °C. To determine which residues are important for the mono- or bi-functional character of KdtA, protein chimeras were constructed using EcKdtA and HiKdtA. These chimeras, which are catalytically active, were characterized by in vitro assays and in vivo complementation. The N-terminal half of KdtA, especially the first 30 amino acid residues, specifies whether one or two Kdo units are transferred to lipid IVA. PMID:20394418

  2. Genetic Contribution of Polymorphisms in Glutathione S-Transferases to Brain Tumor Risk.

    PubMed

    Geng, Peiliang; Li, Jianjun; Wang, Ning; Ou, Juanjuan; Xie, Ganfeng; Sa, Rina; Liu, Chen; Xiang, Lisha; Li, Hongtao; Liang, Houjie

    2016-04-01

    Existing data have shown a major effect of glutathione S-transferase (GST) single-nucleotide polymorphisms on activities of detoxification-related enzymes, and it is the functional importance that leads to extensive research on the association of GST polymorphisms with the risk of developing brain tumor. Previously reported associations, nevertheless, remain inconsistent. This study aimed to reevaluate the association with new information from recent research articles. We weekly searched multiple databases, aiming to cover all studies looking at the associations being examined in this work. Eligibility of studies was evaluated based on predesigned inclusion criteria. To assess the association of GST polymorphisms with brain tumor risk, we calculated genotypic ORs by comparing the number of genotypes between cases and controls. We also detected interstudy heterogeneity, publication bias, and single studies' influence. A total of 13 research articles were identified through databases and hand search. We found significantly elevated risk of brain tumor associated with GSTT1 null status in individuals of European ethnicity (OR 1.46, 95% CI 1.12-1.92). In the analysis of GSTP1 I105V, we observed that Val/Val genotype compared to the Ile/Ile genotype was more prone to a reduced brain tumor risk (OR 0.77, 95% CI 0.64-0.93). Such major effects were similarly seen for GSTP1 A114V (OR 1.14, 95% CI 1.01-1.29 for Val/Val + Ala/Val vs. Ala/Ala). When data were limited to glioma, we found a significant elevation associated with the combination of Val/Val and Ala/Val genotypes (OR 1.18, 95% CI 1.01-1.37). However, no clear association was detected between other polymorphisms investigated and glioma. These statistical data suggest that some of the polymorphisms at GST loci are possibly associated with the genetic risk of brain tumor. PMID:25735248

  3. 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

  4. Interaction of glutathione transferase P1-1 with captan and captafol.

    PubMed

    di Ilio, C; Sacchetta, P; Angelucci, S; Bucciarelli, T; Pennelli, A; Mazzetti, A P; Lo Bello, M; Aceto, A

    1996-07-12

    Glutathione transferase (GST, EC 2.5.1.18) P1-1 was strongly inhibited by captan and captafol in a time- and concentration-dependent manner. The IC50 values for captan and captafol were 5.8 microM and 1.5 microM, respectively. Time-course inactivation of GSTP1-1 by two pesticides was prevented by 3 microM of hexyl-glutathione, but not by methylglutathione. The fact that the inactivated enzyme recovered all the 5,5'-dithiobis(2-nitrobenzoic acid) titrable thiol groups, with concomitant recovery of all its original activity after treatment with 100 microM dithiothreitol, suggested that captan and captafol were able to induce the formation of disulfide bonds. That the inactivation of GSTP1-1 by captan and captafol involves the formation of disulfide bonds between the four cysteinil groups of the enzymes was confirmed by the SDS-PAGE experiments on nondenaturant conditions. In fact, on SDS-PAGE, GSTP1-1 as well as the cys47ala, cys101ala, and cys47ala/cys101ala GSTP1-1 mutants treated with captan and captafol showed several extra bands, with apparent molecular masses higher and lower than the molecular mass of native GSTP1-1 (23.5 kDa), indicating that both intra- and inter-subunit disulfide bonds were formed. These extra bands returned to the native 23.5 kDa band with concomitant restoration of activity when treated with dithiothreitol.

  5. 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

  6. Serum Glutathione S-Transferase P1 1 in Prediction of Cardiac Function

    PubMed Central

    Andrukhova, Olena; Salama, Mohamed; Rosenhek, Raphael; Gmeiner, Matthias; Perkmann, Thomas; Steindl, Johannes; Aharinejad, Seyedhossein

    2012-01-01

    Background Glutathione S-transferase P1 1 (GSTP1) belongs to the multigene isozyme family involved in cellular response to oxidative stress and apoptosis. Our initial retrospective proteomic analysis suggested that GSTP1 is associated with heart failure (HF). Although pro–B-type natriuretic peptide (proBNP) serves currently as a surrogate diagnostic and prognostic parameter in HF patients, its specificity remains uncertain. We hypothesized that GSTP1 might be a useful serum marker in the monitoring of HF patients. Methods and Results Serum GSTP1 and proBNP were prospectively measured in 193 patients subdivided based on their ejection fraction (EF) either in equal-sized quintiles or predefined EF groups >52%, 43%–52%, 33%–42%, 23%–32% and ≤22%. At a cutoff of ≥231 ng/mL, GSTP1 identified HF patients with EF ≤22% with 81% sensitivity and 83% specificity, and at a cutoff of ≥655 pg/mL, proBNP identified the same patient group with 84% sensitivity and 22% specificity. GSTP1 at a ≥126 ng/mL cutoff identified EF ≤42% with 90% sensitivity and 95% specificity, or proBNP at a ≥396 pg/mL cutoff had 97% sensitivity and 20% specificity. In regression analyses, GSTP1, but not proBNP, discriminated between EF ≤42% and EF >42% in HF patients. Conclusions These results suggest that GSTP1 is strongly associated with HF and could serve as a sensitive and specific marker to predict the ventricular function in HF patients. PMID:22385947

  7. 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).

  8. 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

  9. 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

  10. 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

  11. Pharmacological stimulation of brain carnitine palmitoyl-transferase-1 decreases food intake and body weight.

    PubMed

    Aja, Susan; Landree, Leslie E; Kleman, Amy M; Medghalchi, Susan M; Vadlamudi, Aravinda; McFadden, Jill M; Aplasca, Andrea; Hyun, Jayson; Plummer, Erica; Daniels, Khadija; Kemm, Matthew; Townsend, Craig A; Thupari, Jagan N; Kuhajda, Francis P; Moran, Timothy H; Ronnett, Gabriele V

    2008-02-01

    Inhibition of brain carnitine palmitoyl-transferase-1 (CPT-1) is reported to decrease food intake and body weight in rats. Yet, the fatty acid synthase (FAS) inhibitor and CPT-1 stimulator C75 produces hypophagia and weight loss when given to rodents intracerebroventricularly (icv). Thus roles and relative contributions of altered brain CPT-1 activity and fatty acid oxidation in these phenomena remain unclarified. We administered compounds that target FAS or CPT-1 to mice by single icv bolus and examined acute and prolonged effects on feeding and body weight. C75 decreased food intake rapidly and potently at all doses (1-56 nmol) and dose dependently inhibited intake on day 1. Dose-dependent weight loss on day 1 persisted through 4 days of postinjection monitoring. The FAS inhibitor cerulenin produced dose-dependent (560 nmol) hypophagia for 1 day, weight loss for 2 days, and weight regain to vehicle control by day 3. The CPT-1 inhibitor etomoxir (32, 320 nmol) did not alter overall day 1 feeding. However, etomoxir attenuated the hypophagia produced by C75, indicating that CPT-1 stimulation is important for C75's effect. A novel compound, C89b, was characterized in vitro as a selective stimulator of CPT-1 that does not affect fatty acid synthesis. C89b (100, 320 nmol) decreased feeding in mice for 3 days and produced persistent weight loss for 6 days without producing conditioned taste aversion. Similarly, intraperitoneal administration decreased feeding and body weight without producing conditioned taste aversion. These results suggest a role for brain CPT-1 in the regulation of energy balance and implicate CPT-1 stimulation as a pharmacological approach to weight loss.

  12. Key role for a glutathione transferase in multiple-herbicide resistance in grass weeds.

    PubMed

    Cummins, Ian; Wortley, David J; Sabbadin, Federico; He, Zhesi; Coxon, Christopher R; Straker, Hannah E; Sellars, Jonathan D; Knight, Kathryn; Edwards, Lesley; Hughes, David; Kaundun, Shiv Shankhar; Hutchings, Sarah-Jane; Steel, Patrick G; Edwards, Robert

    2013-04-01

    Multiple-herbicide resistance (MHR) in black-grass (Alopecurus myosuroides) and annual rye-grass (Lolium rigidum) is a global problem leading to a loss of chemical weed control in cereal crops. Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to detoxify xenobiotics. In humans, MDR is linked to the overexpression of a pi class glutathione transferase (GSTP1), which has both detoxification and signaling functions in promoting drug resistance. In both annual rye-grass and black-grass, MHR was also associated with the increased expression of an evolutionarily distinct plant phi (F) GSTF1 that had a restricted ability to detoxify herbicides. When the black-grass A. myosuroides (Am) AmGSTF1 was expressed in Arabidopsis thaliana, the transgenic plants acquired resistance to multiple herbicides and showed similar changes in their secondary, xenobiotic, and antioxidant metabolism to those determined in MHR weeds. Transcriptome array experiments showed that these changes in biochemistry were not due to changes in gene expression. Rather, AmGSTF1 exerted a direct regulatory control on metabolism that led to an accumulation of protective flavonoids. Further evidence for a key role for this protein in MHR was obtained by showing that the GSTP1- and MDR-inhibiting pharmacophore 4-chloro-7-nitro-benzoxadiazole was also active toward AmGSTF1 and helped restore herbicide control in MHR black-grass. These studies demonstrate a central role for specific GSTFs in MHR in weeds that has parallels with similar roles for unrelated GSTs in MDR in humans and shows their potential as targets for chemical intervention in resistant weed management.

  13. Variability of glutathione S-transferase isoenzyme patterns in matched normal and cancer human breast tissue.

    PubMed Central

    Kelley, M K; Engqvist-Goldstein, A; Montali, J A; Wheatley, J B; Schmidt, D E; Kauvar, L M

    1994-01-01

    The determination of GST levels in blood has been proposed to a marker of tumour burden in general, whereas level of the P1 isoenzyme has been identified as a prognostic factor for breast-cancer patients receiving no adjuvant chemotherapy. Particular glutathione S-transferase (GST) isoenzymes differ in their substrate specificity, however, and their presence or absence might therefore account for the resistance of tumours to particular chemotherapeutic drugs, as already established for cultured cell lines. Determination of the GST isoenzyme profile of a cancer tissue could have prognostic value in the selection of treatment if the levels of expression/activity show a degree of variation comparable with that exhibited by actual patient responses. Using reversed-phase h.p.l.c. to quantify affinity-isolated GSTs, we have analysed full isoenzyme profiles in the first large sample of matched normal and cancer human tissues (18 breast-cancer patients). In no patients did the tumour tissues express any isoenzymes that were not found in normal breast tissue. In addition to the GSTs, another enzyme, identified as enoyl-CoA isomerase, was regularly found in breast tissue cytosol following elution from a hexyl-glutathione affinity column. In most cases, the average level of GST was substantially elevated in the cancer tissues above the levels in normal breast tissue from the same patient. Furthermore, the relative levels of the isoenzymes were substantially more variable in the cancer samples than in the normal breast tissue, providing a plausible mechanism for the well established variable response to treatment. Images Figure 1 Figure 3 PMID:7818489

  14. Overexpression of GalNAc-transferase GalNAc-T3 promotes pancreatic cancer cell growth.

    PubMed

    Taniuchi, K; Cerny, R L; Tanouchi, A; Kohno, K; Kotani, N; Honke, K; Saibara, T; Hollingsworth, M A

    2011-12-01

    O-linked glycans of secreted and membrane-bound proteins have an important role in the pathogenesis of pancreatic cancer by modulating immune responses, inflammation and tumorigenesis. A critical aspect of O-glycosylation, the position at which proteins are glycosylated with N-acetyl-galactosamine on serine and threonine residues, is regulated by the substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-Ts). Thus, GalNAc-Ts regulate the first committed step in O-glycosylated protein biosynthesis, determine sites of O-glycosylation on proteins and are important for understanding normal and carcinoma-associated O-glycosylation. We have found that one of these enzymes, GalNAc-T3, is overexpressed in human pancreatic cancer tissues and suppression of GalNAc-T3 significantly attenuates the growth of pancreatic cancer cells in vitro and in vivo. In addition, suppression of GalNAc-T3 induces apoptosis of pancreatic cancer cells. Our results indicate that GalNAc-T3 is likely involved in pancreatic carcinogenesis. Modification of cellular glycosylation occurs in nearly all types of cancer as a result of alterations in the expression levels of glycosyltransferases. We report guanine the nucleotide-binding protein, α-transducing activity polypeptide-1 (GNAT1) as a possible substrate protein of GalNAc-T3. GalNAc-T3 is associated with O-glycosylation of GNAT1 and affects the subcellular distribution of GNAT1. Knocking down endogenous GNAT1 significantly suppresses the growth/survival of PDAC cells. Our results imply that GalNAc-T3 contributes to the function of O-glycosylated proteins and thereby affects the growth and survival of pancreatic cancer cells. Thus, substrate proteins of GalNAc-T3 should serve as important therapeutic targets for pancreatic cancers.

  15. The Stereochemical Course of 4-Hydroxy-2-nonenal Metabolism by Glutathione S-Transferases*S⃞

    PubMed Central

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

    2008-01-01

    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

  16. 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.

  17. 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

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

    PubMed

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

    2015-12-01

    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.

  19. Glutathione S-transferase M1 null genotype related to poor prognosis of colorectal cancer.

    PubMed

    Yan, Shushan; Wang, Zengfang; Wang, Zengyan; Duan, Quanhong; Wang, Xiaochen; Li, Jun; Sun, Beicheng

    2016-08-01

    Published studies showed controversial findings about the relationship between glutathione S-transferase M1 (GSTM1) null genotype and clinical outcomes of patients with colorectal cancer. We performed a meta-analysis to quantitatively assess the association between GSTM1 null genotype and prognosis of patients with colorectal cancer. We systematically searched Pubmed, Embase, and Web of Science to identify prospective or retrospective cohort studies assessing the association of GSTM1 null genotype with overall survival (OS) or disease-free survival (DFS) in colorectal cancer. The hazard ratios (HRs) and 95 % confidence intervals (95 % CIs) were used to assess the association of GSTM1 null genotype with OS or DFS. Finally, 15 studies from 14 publications with 4326 colorectal cancer patients were included into the meta-analysis. There was no heterogeneity in the meta-analysis relating OS (I (2) = 0 %) and DFS (I (2) = 0 %). Overall, GSTM1 null genotype was significantly associated with poor OS in patients with colorectal cancer (HR = 1.18, 95 % CI 1.07-1.30, P = 0.001). In addition, GSTM1 null genotype was also significantly associated with poor DFS in patients with colorectal cancer (HR = 1.15, 95 % CI 1.03-1.28, P = 0.015). No obvious risk of publication bias was observed. GSTM1 null genotype is significantly associated with poor OS and DFS in patients with colorectal cancer, which suggests that GSTM1 null genotype confers poor effect on the prognosis of colorectal cancer.

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

    PubMed

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

    2015-06-01

    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.

  1. 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

  2. Effect of protein-calorie malnutrition on cytochromes P450 and glutathione S-transferase.

    PubMed

    Zhang, W; Parentau, H; Greenly, R L; Metz, C A; Aggarwal, S; Wainer, I W; Tracy, T S

    1999-01-01

    Protein-calorie malnutrition (PCM) can develop both from inadequate food intake and as a consequence of diseases such as cancer and AIDS. Several studies have shown that PCM can alter drug clearance but little information is available on the effect of PCM on individual cytochrome P450 isoforms and phase II conjugation enzymes. The aim of the present study was to begin a systematic evaluation of the effect of PCM on the activity of individual drug metabolizing enzymes in a rat model of PCM. Control and PCM rats received isocaloric diets which contained either 21% or 5% (deficient) protein. After 3 weeks, the animals were sacrificed and microsomal and cytosolic fractions prepared. Ethoxyresorufin O-deethylation (EROD), chlorzoxazone 6-hydroxylation, dextromethorphan N- and O-demethylation and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation were used as measures of CYP1A, CYP2E1, CYP3A2, CYP2D1 and glutathione S-transferase (GST) activity, respectively. Additionally, NADPH-cytochrome P450 reductase activity was measured in the liver microsomes. PCM significantly reduced the maximum velocity (Vmax) of all model reactions studied. However, differential effects were observed with respect to K(m) values of the reactions. The K(m) values for EROD and dextromethorphan N-demethylation were significantly increased in PCM animals, whereas the K(m) values for chlorzoxazone 6-hydroxylation and dextromethorphan O-demethylation were decreased. In contrast, the K(m) value for CDNB conjugation was unchanged. When NADPH-cytochrome P450 reductase activity was compared, a 29% reduction in reductase activity was noted in PCM animals as compared to controls. Thus, it appears that PCM decreases the overall activity of certain phase I and phase II metabolism enzymes in rat liver while exhibiting differential effects on K(m). Furthermore, this reduction in activity may be due in part to diminished activity of cytochrome P450 reductase.

  3. Localization of epidermal sphingolipid synthesis and serine palmitoyl transferase activity: alterations imposed by permeability barrier requirements.

    PubMed

    Holleran, W M; Gao, W N; Feingold, K R; Elias, P M

    1995-01-01

    Sphingolipids, the predominant lipid species in mammalian stratum corneum play, a central role in permeability barrier homeostatis. Prior studies have shown that the epidermis synthesizes abundant sphingolipids, a process regulated by barrier requirements, and that inhibition of sphingolipid synthesis interferes with barrier homeostasis. To investigate further the relationship between epidermal sphingolipid metabolism and barrier function, we localized sphingolipid synthetic activity in murine epidermis under basal conditions, and following acute (acetone treatment) or chronic (essential fatty acid deficiency, EFAD) barrier perturbation, using dithiothreitol and/or the staphylococcal epidermolytic toxin to isolate the lower from the outer epidermis. Under basal conditions, both the activity of serine palmitoyl transferase (SPT), the rate-limiting enzyme of sphingolipid synthesis, and the rates of 3H-H2O incorporation into sphingolipids were nearly equivalent in the lower and the outer epidermis. Following acute barrier perturbation, SPT activity increased significantly in both the lower (35%; P < 0.05) and the outer epidermal layers (60%; P < 0.01). The rates of 3H-H2O incorporation into each major sphingolipid family, including ceramides, glucosylceramides and sphingomyelin, increased significantly in both the lower and the outer epidermis of treated flanks after acute barrier disruption. Finally, SPT activity was modestly elevated (20%; P < 0.01) in the lower but not in the outer epidermis of EFAD animals. These studies demonstrate the ability of both lower and outer epidermal cells to generate sphingolipids, and that permeability barrier homeostatic mechanisms appear to differentially regulate SPT activity and sphingolipid synthesis in the lower and the outer epidermis in response to acute and chronic barrier perturbation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7598529

  4. 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

  5. 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

  6. Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

    PubMed Central

    Huenchuguala, Sandro; Muñoz, Patricia; Zavala, Patricio; Villa, Mónica; Cuevas, Carlos; Ahumada, Ulises; Graumann, Rebecca; Nore, Beston F; Couve, Eduardo; Mannervik, Bengt; Paris, Irmgard; Segura-Aguilar, Juan

    2014-01-01

    U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit 3H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction. PMID:24434817

  7. 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

  8. 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.

  9. 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

  10. 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

  11. Functional characterization of alpha-class glutathione s-transferases from the Turkey (meleagris gallopavo).

    PubMed

    Kim, Ji Eun; Bunderson, Brett R; Croasdell, Amanda; Coulombe, Roger A

    2011-11-01

    Six Alpha-class glutathione S-transferase (GST) subunits were cloned from domestic turkey livers, which are one of the most susceptible animals known to the carcinogenic mycotoxin aflatoxin B₁. In most animals, GST dysfunction is a risk factor for susceptibility toward AFB₁, and we have shown that turkeys lack GSTs with affinity toward the carcinogenic intermediate exo-aflatoxin B(1)-8-9-epoxide (AFBO). Conversely, mice are resistant to AFB₁ carcinogenesis, due to high constitutive expression of mGSTA3 that has high affinity toward AFBO. When expressed in Escherichia coli, all six tGSTA subunits possessed conjugating activities toward substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), and cumene hydroperoxide (CHP) with tGSTA1.2 appearing most active. Interestingly, tGSTA1.1, which lacks one of the four Alpha-class signature motifs, possessed enzymatic activities toward all substrates. All had comparable activities toward AFBO conjugation, an activity absent in turkey liver cytosols. E. coli-expressed mGSTA3 conjugated AFBO with more than 3-fold greater activity than that of tGSTAs and had higher activity toward GST prototype substrates. Mouse hepatic cytosols had approximately 900-fold higher catalytic activity toward AFBO compared with those from turkey. There was no apparent amino acid profile in tGSTAs that might correspond to specificity toward AFBO, although tGSTA1.2, which had slightly higher AFBO-trapping ability, shared Tyr¹⁰⁸ with mGSTA3, a residue postulated to be critical for AFBO trapping activity in mammalian systems. The observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are silenced by one or more regulatory mechanisms.

  12. Glutathione-S-transferases in lung and sputum specimens, effects of smoking and COPD severity

    PubMed Central

    Harju, Terttu; Mazur, Witold; Merikallio, Heta; Soini, Ylermi; Kinnula, Vuokko L

    2008-01-01

    Background Oxidative stress plays a potential role in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). Glutathione S-transferases (GSTs) detoxify toxic compounds in tobacco smoke via glutathione-dependent mechanisms. Little is known about the regulation and expression of GSTs in COPD lung and their presence in airway secretions. Methods GST alpha, pi and mu were investigated by immunohistochemistry in 72 lung tissue specimens and by Western analysis in total lung homogenates and induced sputum supernatants from non-smokers, smokers and patients with variable stages of COPD severity. Results GST alpha was expressed mainly in the airway epithelium. The percentage of GST alpha positive epithelial cells was lower in the central airways of patients with very severe (Stage IV) COPD compared to mild/moderate COPD (p = 0.02). GST alpha by Western analysis was higher in the total lung homogenates in mild/moderate COPD compared to cases of very severe disease (p < 0.001). GST pi was present in airway and alveolar epithelium as well as in alveolar macrophages. GST mu was expressed mainly in the epithelium. Both GST alpha and pi were detectable in sputum supernatants especially in patients with COPD. Conclusion This study indicates the presence of GST alpha and pi especially in the epithelium and sputum supernatants in mild/moderate COPD and low expression of GST alpha in the epithelium in cases of very severe COPD. The presence of GSTs in the airway secretions points to their potential protective role both as intracellular and extracellular mediators in human lung. PMID:19077292

  13. Glutathione S-Transferase Polymorphisms, Passive Smoking, Obesity, and Heart Rate Variability in Nonsmokers

    PubMed Central

    Probst-Hensch, Nicole M.; Imboden, Medea; Dietrich, Denise Felber; Barthélemy, Jean-Claude; Ackermann-Liebrich, Ursula; Berger, Wolfgang; Gaspoz, Jean-Michel; Schwartz, Joel

    2008-01-01

    Background Disturbances of heart rate variability (HRV) may represent one pathway by which second-hand smoke (SHS) and air pollutants affect cardiovascular morbidity and mortality. The mechanisms are poorly understood. Objectives We investigated the hypothesis that oxidative stress alters cardiac autonomic control. We studied the association of polymorphisms in oxidant-scavenging glutathione S-transferase (GST) genes and their interactions with SHS and obesity with HRV. Methods A total of 1,133 nonsmokers > 50 years of age from a population-based Swiss cohort underwent ambulatory 24-hr electrocardiogram monitoring and reported on lifestyle and medical history. We genotyped GSTM1 and GSTT1 gene deletions and a GSTP1 (Ile105Val) single nucleotide polymorphism and analyzed genotype–HRV associations by multiple linear regressions. Results Homozygous GSTT1 null genotypes exhibited an average 10% decrease in total power (TP) and low-frequency-domain HRV parameters. All three polymorphisms modified the cross-sectional associations of HRV with SHS and obesity. Homozygous GSTM1 null genotypes with > 2 hr/day of SHS exposure exhibited a 26% lower TP [95% confidence interval (CI), 11 to 39%], versus a reduction of −5% (95% CI, −22 to 17%) in subjects with the gene and the same SHS exposure compared with GSTM1 carriers without SHS exposure. Similarly, obese GSTM1 null genotypes had, on average, a 22% (95% CI, 12 to 31%) lower TP, whereas with the gene present obesity was associated with only a 3% decline (95% CI, −15% to 10%) compared with nonobese GSTM1 carriers. Conclusions GST deficiency is associated with significant HRV alterations in the general population. Its interaction with SHS and obesity in reducing HRV is consistent with an impact of oxidative stress on the autonomous nervous system. PMID:19057702

  14. Properties of a Maize Glutathione S-Transferase That Conjugates Coumaric Acid and Other Phenylpropanoids.

    PubMed Central

    Dean, J. V.; Devarenne, T. P.; Lee, I. S.; Orlofsky, L. E.

    1995-01-01

    A glutathione S-transferase (GST) enzyme from corn (Zea mays L. Pioneer hybrid 3906) that is active with p-coumaric acid and other unsaturated phenylpropanoids was purified approximately 97-fold and characterized. The native enzyme appeared to be a monomer with a molecular mass of approximately 30 kD and an apparent isoelectric point at pH 5.2. The enzyme had a pH optimum between 7.5 and 8.0 and apparent Km values of 4.4 and 1.9 mM for reduced glutathione (GSH) and p-coumaric acid, respectively. In addition to p-coumaric acid, the enzyme was also active with o-coumaric acid, m-coumaric acid, trans-cinnamic acid, ferulic acid, and coniferyl alcohol. In addition to GSH, the enzyme could also utilize cysteine as a sulfhydryl source. The enzyme activity measured when GSH and trans-cinnamic acid were used as substrates was enhanced 2.6- and 5.2-fold by the addition of 50 [mu]M p-coumaric acid and 7-hydroxycoumarin, respectively. 1H- and 13C-nuclear magnetic resonance spectroscopic analysis of the conjugate revealed that the enzyme catalyzed the addition of GSH to the olefinic double bond of p-coumaric acid. Based on the high activity and the substrate specificity of this enzyme, it is possible that this enzyme may be involved in the in vivo conjugation of a number of unsaturated phenylpropanoids. PMID:12228522

  15. 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.

  16. Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity.

    PubMed

    Ahmad, Shabbir; Thulasingam, Madhuranayaki; Palombo, Isolde; Daley, Daniel O; Johnson, Kenneth A; Morgenstern, Ralf; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

    2015-10-01

    Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA4 forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755-1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometry of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.

  17. A role for glutathione transferase Omega 1 (GSTO1-1) in the glutathionylation cycle.

    PubMed

    Menon, Deepthi; Board, Philip G

    2013-09-01

    The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

  18. Resistance to acetaminophen-induced hepatotoxicity in glutathione S-transferase Mu 1-null mice.

    PubMed

    Arakawa, Shingo; Maejima, Takanori; Fujimoto, Kazunori; Yamaguchi, Takashi; Yagi, Masae; Sugiura, Tomomi; Atsumi, Ryo; Yamazoe, Yasushi

    2012-01-01

    We investigated the role of glutathione S-transferases Mu 1 (GSTM1) in acetaminophen (APAP)-induced hepatotoxicity using Gstm1-null mice. A single oral administration of APAP resulted in a marked increase in plasma alanine aminotransferase accompanied by hepatocyte necrosis 24 hr after administration in wild-type mice, but its magnitude was unexpectedly attenuated in Gstm1-null mice. Therefore, it is suggested that Gstm1-null mice are resistant to APAP-induced hepatotoxicity. To examine the mechanism of this resistance in Gstm1-null mice, we measured phosphorylation of c-jun N-terminal kinase (JNK), which mediates the signal of APAP-induced hepatocyte necrosis, by Western blot analysis 2 and 6 hr after APAP administration. A marked increase in phosphorylated JNK was observed in wild-type mice, but the increase was markedly suppressed in Gstm1-null mice. Therefore, it is suggested that suppressed phosphorylation of JNK may be a main mechanism of the resistance to APAP-induced hepatotoxicity in Gstm1-null mice, although other possibilities of the mechanism cannot be eliminated. Additionally, phosphorylation of glycogen synthase kinase-3β and mitogen-activated protein kinase kinase 4, which are upstream kinases of JNK in APAP-induced hepatotoxicity, were also suppressed in Gstm1-null mice. A decrease in liver total glutathione 2 hr after APAP administration, which is an indicator for exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of APAP, were similar in wild-type and Gstm1-null mice. In conclusion, Gstm1-null mice are considered to be resistant to APAP-induced hepatotoxicity perhaps by the suppression of JNK phosphorylation. This study indicates the novel role of GSTM1 as a factor mediating the cellular signal for APAP-induced hepatotoxicity.

  19. Characterization and functional analysis of four glutathione S-transferases from the migratory locust, Locusta migratoria.

    PubMed

    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 Ni(2+)-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 (Cu(2+) and Cd(2+)). 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. Glutathione S-Transferase Regulation in Calanus finmarchicus Feeding on the Toxic Dinoflagellate Alexandrium fundyense.

    PubMed

    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

  8. 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

  9. 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

  10. Rescue of Drosophila Melanogaster l(2)35Aa lethality is only mediated by polypeptide GalNAc-transferase pgant35A, but not by the evolutionary conserved human ortholog GalNAc-transferase-T11.

    PubMed

    Bennett, Eric P; Chen, Ya-Wen; Schwientek, Tilo; Mandel, Ulla; Schjoldager, Katrine ter-Borch Gram; Cohen, Stephen M; Clausen, Henrik

    2010-05-01

    The Drosophila l(2)35Aa gene encodes a UDP-N-acetylgalactosamine: Polypeptide N-acetylgalactosaminyltransferase, essential for embryogenesis and development (J. Biol. Chem. 277, 22623-22638; J. Biol. Chem. 277, 22616-22). l(2)35Aa, also known as pgant35A, is a member of a large evolutionarily conserved family of genes encoding polypeptide GalNAc-transferases. Phylogenetic and functional analyses have proposed that subfamilies of orthologous GalNAc-transferase genes are conserved in species, suggesting that they serve distinct functions in vivo. Based on sequence alignments, pgant35A and human GALNT11 are thought to belong to a distinct subfamily. Recent in vitro studies have shown that pgant35A and pgant7, encoding enzymes from different subfamilies, prefer different acceptor substrates, whereas the orthologous pgant35A and human GALNT11 gene products possess, 1) conserved substrate preferences and 2) similar acceptor site preferences in vitro. In line with the in vitro pgant7 studies, we show that l(2)35Aa lethality is not rescued by ectopic pgant7 expression. Remarkably and in contrast to this observation, the human pgant35A ortholog, GALNT11, was shown not to support rescue of the l(2)35Aa lethality. By use of genetic "domain swapping" experiments we demonstrate, that lack of rescue was not caused by inappropriate sub-cellular targeting of functionally active GalNAc-T11. Collectively our results show, that fly embryogenesis specifically requires functional pgant35A, and that the presence of this gene product during fly embryogenesis is functionally distinct from other Drosophila GalNAc-transferase isoforms and from the proposed human ortholog GALNT11.

  11. Regulatory and functional interactions of plant growth regulators and plant glutathione S-transferases (GSTs).

    PubMed

    Moons, Ann

    2005-01-01

    Plant glutathioneS-transferases (GSTs) are a heterogeneous superfamily of multifunctional proteins, grouped into six classes. The tau (GSTU) and phi (GSTF) class GSTs are the most represented ones and are plant-specific, whereas the smaller theta (GSTT) and zeta (GSTZ) classes are also found in animals. The lambda GSTs (GSTL) and the dehydroascorbate reductases (DHARs) are more distantly related. Plant GSTs perform a variety of pivotal catalytic and non-enzymatic functions in normal plant development and plant stress responses, roles that are only emerging. Catalytic functions include glutathione (GSH)-conjugation in the metabolic detoxification of herbicides and natural products. GSTs can also catalyze GSH-dependent peroxidase reactions that scavenge toxic organic hydroperoxides and protect from oxidative damage. GSTs can furthermore catalyze GSH-dependent isomerizations in endogenous metabolism, exhibit GSH-dependent thioltransferase safeguarding protein function from oxidative damage and DHAR activity functioning in redox homeostasis. Plant GSTs can also function as ligandins or binding proteins for phytohormones (i.e., auxins and cytokinins) or anthocyanins, thereby facilitating their distribution and transport. Finally, GSTs are also indirectly involved in the regulation of apoptosis and possibly also in stress signaling. Plant GST genes exhibit a diversity of expression patterns during biotic and abiotic stresses. Stress-induced plant growth regulators (i.e., jasmonic acid [JA], salicylic acid [SA], ethylene [ETH], and nitric oxide [NO] differentially activate GST gene expression. It is becoming increasingly evident that unique combinations of multiple, often interactive signaling pathways from various phytohormones and reactive oxygen species or antioxidants render the distinct transcriptional activation patterns of individual GSTs during stress. Underestimated post-transcriptional regulations of individual GSTs are becoming increasingly evident and roles

  12. Isolation, cloning and large scale expression of glutathione-S-transferase (GST) protein of Polymyxa betae.

    PubMed

    Safarpour, H; Safarnejad, M R

    2012-01-01

    The plasmodiophoromycete Polymyxa betae, an obligate parasite of sugar-beet roots, is a natural vector of Beet necrotic yellow vein virus (BNYVV). To develop protein based diagnosis for any pathogenic agents including P. betae, a specific immunogenic protein has to be prepared. The glutathione-S-transferase (GST) is expressed in all the morphologically different stages of the pathogen's life cycle, and then it is a good candidate as an immunogenic agent for developing of specific antibodies and diagnostic purposes. The present study describes isolation, cloning and large scale expression and purification of P. betae GST protein. For this aim, total RNA was initially isolated from infected plants and corresponding cDNA was constructed by using reverse transcriptase and oligo-dT primer as well as mRNA as a template. The gene encoding GST was isolated and PCR-amplified from the synthesized cDNA by using specific primers. The amplified fragments were preliminary cloned into pTZ57R/T cloning vector. Intact clone containing right sequence was selected after digestion, PCR amplification and subsequent sequencing analysis. Next, GST encoding region having right sequence was recovered and sub-cloned into pET28a bacterial expression vector. Large scale expression of recombinant protein was performed in BL21-de3 strain of E. coli and purification was carried out under native situation through Immobolized metal ion affinity chromatography (IMAC) in column containing Ni-NTA agarose beads. Successful expression and purification steps were confirmed by SDS-PAGE followed by western blotting analysis. These results confirmed the high purity and integrity of GST protein which was around 21 kDa. Generally, the total yield of the purified protein in the culture medium was estimated at around 3.5 mg/mL. After purification, a major part of the purified proteins was precipitated identified as excess GST. To improve the solubility, the final concentration of purified protein was reduced

  13. 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

  14. Can we use serum gamma-glutamyl transferase levels to predict early mortality in stroke?

    PubMed Central

    Akinci, Emine; Doğan, Nurettin Özgür; Gümüş, Haluk; Akilli, Nazire belgin

    2014-01-01

    Objective: Serum gamma-glutamyl transferase (GGT) is a marker for alcohol consumption and hepatobiliary diseases. There are reports on the prognostic role of GGT in coronary artery diseases and stroke. The aim of our study was to identify the potential differences in GGT levels in different types of stroke, and to evaluate the correlation between GGT and 30-day mortality. Method: Patients diagnosed with stroke in emergency department between 01.01.2010 and 30.12.2012 was included in the study. Imaging techniques were used to distinguish between hemorrhagic and ischemic stroke. Ischemic strokes were further classified as either atherosclerotic/lacunar or embolic. Parameters including age, gender, vital signs (systolic and diastolic blood pressure), comorbid diseases (HT, DM, CAD, smoking and alcohol consumption), used medications, previous history of stroke, NIHSS score at the time of admission to emergency department, laboratory parameters (glucose, white blood cell count, hemoglobin, platelet, total cholesterol, creatinine) and duration of hospitalization were recorded. Death records were obtained from patients’ medical records. Results: One thousand eighty six patients were included in the study. GGT levels were not significantly different between ischemic and hemorrhagic strokes (p=0.435). On the other hand, GGT levels in embolic strokes were significantly higher compared to atherosclerotic/lacunar strokes (p=0.001). GGT levels [median 24.50 (16.00-43.00)] in Intensive Care Unit patients were significantly higher compared to GGT level [22.00 (15.00-34.25)] in admitted to service beds patients (p=0.015). Median GGT level of deceased patients was 24.00 (16.00-41.25) and median GGT level of alive patients was 22.00 (15.00-35.00). GGT level of deceased patients was significantly higher compared to GGT levels of alive patients (p=0.048). Conclusion: There was no difference in GGT levels between ischemic and hemorrhagic strokes; however, GGT levels in embolic

  15. 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

  16. 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

  17. Glutathione S-Transferase Gene Polymorphisms and Treatment Outcome in Cervical Cancer Patients under Concomitant Chemoradiation

    PubMed Central

    Abbas, Mohammad; Kushwaha, Vandana Singh; Srivastava, Kirti; Banerjee, Monisha

    2015-01-01

    Purpose Cisplatin based concomitant chemoradiation (CRT) is the standard treatment for locally advanced cervical cancer (CC). Glutathione S-transferase (GST), a phase II antioxidant enzyme is induced by oxidative stress generated by drugs and reactive oxidants. The present study was undertaken to evaluate the association of GSTM1, T1 and P1 polymorphisms with the outcome of CRT treatment in CC patients. Methods A total of 227 cervical cancer patients with stages IIB-IIIB treated with the same chemoradiotherapy regimen were enrolled and genotyped for GSTM1, T1 and P1 gene polymorphisms by multiplex polymerase chain reaction (mPCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Overall survival was evaluated using Kaplan-Meier survival function and Cox proportional hazards model. All data were analyzed using SPSS (version 21.0). Results Stratified analysis showed that GSTM1 null (M1-) genotype was associated with a significantly better survival among patients with stage IIB cervical cancer (log-rank P = 0.004) than cases with stage IIIA/IIIB. Death and recurrence were significantly higher in patients with GSTM1 present genotype (M1+) (P = 0.037 and P = 0.003 respectively) and those with M1- showed reduced hazard of death with an adjusted hazard ratio ‘HR’ of 0.47 (95% CI, 0.269–0.802, P = 0.006). Women with M1- genotype as well as in combination with GSTT1 null (T1-), GSTP1 (AG+GG) and GSTT1 null/GSTP1 (AG+GG) showed better survival and also reduced risk of death (HR = 0.31, P = 0.016; HR = 0.45, P = 0.013; HR = 0.31, P = 0.02 respectively). Conclusions To the best of our knowledge, this is the first study to correlate the association of GSTM1, T1 and P1 gene polymorphisms with treatment outcome of CRT treated CC patients. Our results suggested that individuals with GSTM1 null genotype and in combination with GSTT1 null and GSTP1 (AG+GG) had a survival advantage. Such genetic studies may provide prognostic information in CRT treated CC patients

  18. 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.

  19. Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice.

    PubMed

    Hojjati, Mohammad Reza; Li, Zhiqiang; Jiang, Xian-Cheng

    2005-10-15

    Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, and all these functions may have an impact on atherosclerotic development. Serine palmitoyl-CoA transferase (SPT) is the key enzyme in sphingolipid biosynthesis. To evaluate in vivo SPT activity and its role in sphingolipid metabolism, we applied homologous recombination to embryonic stem cells, producing mice with long chain base 1 (Sptlc1) and long chain base 2 (Sptlc2), two subunits of SPT, gene deficiency. Homozygous Sptlc11 and Sptlc2 mice are embryonic lethal, whereas heterozygous versions of both animals (Sptlc1(+/-), Sptlc2(+/-)) are healthy. Analysis showed that, compared with WT mice, Sptlc1(+/-) and Sptlc2(+/-) mice had: (1) decreased liver Sptlc1 and Sptlc2 mRNA by 44% and 57% (P<0.01 and P<0.0001, respectively); (2) decreased liver Sptlc1 mass by 50% and Sptlc2 mass by 70% (P<0.01 and P<0.01, respectively), moreover, Sptlc1 mass decreased by 70% in Sptlc2(+/-) mouse liver, while Sptlc2 mass decreased by 53% in Sptlc1(+/-) mouse liver (P<0.001 and P<0.01, respectively); (3) decreased liver SPT activity by 45% and 60% (P<0.01, respectively); (4) decreased liver ceramide (22% and 39%, P<0.05 and P<0.01, respectively) and sphingosine levels (22% and 31%, P<0.05 and P<0.01, respectively); (5) decreased plasma ceramide (45% and 39%, P<0.01, respectively), sphingosine-1-phosphate (31% and 32%, P<0.01, respectively) and sphingosine levels (22.5% and 25%, P<0.01, respectively); (6) dramatically decreased plasma lysosphingomyelin (17-fold and 16-fold, P<0.0001, respectively); and (7) no change of plasma sphingomyelin, triglyceride, total cholesterol, phospholipids, and liver sphingomyelin levels. These results indicated that both Sptlc1 and Sptlc2 interactions are necessary for SPT activity in vivo, and that SPT activity directly influences plasma sphingolipid levels. Furthermore, manipulation of SPT activity might well influence the

  20. 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

  1. Cloning and characterization of a biotic-stress-inducible glutathione transferase from Phaseolus vulgaris.

    PubMed

    Chronopoulou, Evangelia; Madesis, Panagiotis; Tsaftaris, Athanasios; Labrou, Nikolaos E

    2014-01-01

    Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its K m for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus

  2. A Mannosyl Transferase Required for Lipopolysaccharide Inner Core Assembly in Rhizobium leguminosarum

    PubMed Central

    Kanipes, Margaret I.; Ribeiro, Anthony A.; Lin, Shanhua; Cotter, Robert J.; Raetz, Christian R. H.

    2008-01-01

    The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo2-lipid IVA. LpcC containing an N-terminal His6 tag was assayed using GDP-mannose as the donor and Kdo2-[4′-32P]lipid IVA as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo2-[4′-32P]lipid IVA. A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IVA or Kdo-lipid IVA. The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the α-anomeric configuration) for the glycosylation of Kdo2-[4′-32P]lipid IVA. Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner L-glycero-D-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo2-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells. PMID:12591937

  3. Association of serum gamma-glutamyl transferase with treatment outcome in chronic hepatitis B patients

    PubMed Central

    Huang, Rui; Yang, Chen-Chen; Liu, Yong; Xia, Juan; Su, Ran; Xiong, Ya-Li; Wang, Gui-Yang; Sun, Zhen-Hua; Yan, Xiao-Min; Lu, Shan; Wu, Chao

    2015-01-01

    AIM: To investigate the association of serum gamma-glutamyl transferase (GGT) levels with chronic hepatitis B infection and hepatitis B e antigen (HBeAg) seroconversion. METHODS: A retrospective study was performed on clinical data collected from patients who had been positive for hepatitis B surface antigen for > 6 mo and who were antiviral-treatment naïve (n = 215) attending the Hepatitis Clinic at Nanjing Drum Tower Hospital between August 2010 and December 2013. Healthy individuals without liver disease (n = 83) were included as controls. Patients were categorized into four groups based on disease status as recommended by the European Association for the Study of the Liver: immune tolerance (IT; n = 47), HBeAg-positive hepatitis (EPH; n = 93), HBeAg-negative hepatitis (ENH; n = 20), and inactive carrier (IC; n = 55). Prediction of complete response (CR) based on serum GGT was also examined in EPH patients (n = 33) treated for 48 wk with nucleos(t)ide analogue (NA) therapy, including lamivudine plus adefovir combination therapy (n = 20) or entecavir monotherapy (n = 13). CR was defined as a serum hepatitis B virus DNA level < 500 copies/mL and HBeAg seroconversion by 48 wk of treatment. RESULTS: Serum GGT levels were significantly increased in EPH and ENH patients relative to the IT, IC, and healthy control groups (P < 0.01 for all). However, no significant difference in serum GGT levels was found between the EPH and ENH groups. Baseline serum GGT levels were significantly higher in patients who achieved CR (7/33; 21.2%) compared to patients in the non-CR group (26/33; 78.8%; P = 0.011). In addition, the decline in serum GGT was greater in CR patients compared to non-CR patients after 24 wk and 48 wk of treatment (P = 0.012 and P = 0.008, respectively). The receiver operating characteristic curve yielded a sensitivity of 85.71% and a specificity of 61.54% at a threshold value of 0.89 times the upper limit of normal for baseline serum GGT in the prediction of CR

  4. Dietary Patterns and Serum Gamma-Glutamyl Transferase in Japanese Men and Women

    PubMed Central

    Nanri, Hinako; Hara, Megumi; Nishida, Yuichiro; Shimanoe, Chisato; Nakamura, Kazuyo; Higaki, Yasuki; Imaizumi, Takeshi; Taguchi, Naoto; Sakamoto, Tatsuhiko; Horita, Mikako; Shinchi, Koichi; Kokaze, Akatsuki; Tanaka, Keitaro

    2015-01-01

    Background Although specific foods and nutrients have been examined as potential determinants of serum gamma-glutamyl transferase (GGT) concentrations, the relationship between dietary patterns and GGT remains unknown. The present cross-sectional study aimed to determine relationships between dietary patterns and GGT concentrations, and the effects of lifestyle factors on GGT. Methods Relationships between dietary patterns and GGT were analyzed in 9803 Japanese individuals (3723 men and 6080 women age 40–69 years) without a history of liver diseases or elevated serum aminotransferase. We examined major dietary patterns by factor analysis of 46 items determined from a validated, short food frequency questionnaire. Results We defined dietary patterns as healthy, Western, seafood, bread, and dessert. The healthy pattern was inversely related to GGT in men (odds ratio [OR] for highest vs lowest quartile, 0.72; 95% confidence interval [CI], 0.57–0.92; P < 0.01 for trend) and women (OR 0.82; 95% CI, 0.66–1.0; P = 0.05 for trend), whereas the seafood pattern was positively related to GGT in men (OR 1.27; 95% CI, 1.01–1.61; P = 0.03 for trend) and women (OR 1.21; 95% CI, 0.98–1.49; P = 0.05 for trend). Male-specific inverse associations with GGT were found for bread and dessert patterns (OR 0.63; 95% CI, 0.50–0.80 and OR 0.53; 95% CI, 0.41–0.68, respectively; P < 0.01 for both trends). Seafood or bread patterns and alcohol consumption significantly interacted with GGT in men (P = 0.03 and <0.01 for interaction, respectively) and between the dessert pattern and body mass index or smoking habit in women (P = 0.03 and <0.01, respectively, for interaction). Conclusions Dietary patterns may be important determinants of GGT, and their possible clinical implications warrant further investigation. PMID:25787241

  5. 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

  6. Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice.

    PubMed

    Hojjati, Mohammad Reza; Li, Zhiqiang; Jiang, Xian-Cheng

    2005-10-15

    Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, and all these functions may have an impact on atherosclerotic development. Serine palmitoyl-CoA transferase (SPT) is the key enzyme in sphingolipid biosynthesis. To evaluate in vivo SPT activity and its role in sphingolipid metabolism, we applied homologous recombination to embryonic stem cells, producing mice with long chain base 1 (Sptlc1) and long chain base 2 (Sptlc2), two subunits of SPT, gene deficiency. Homozygous Sptlc11 and Sptlc2 mice are embryonic lethal, whereas heterozygous versions of both animals (Sptlc1(+/-), Sptlc2(+/-)) are healthy. Analysis showed that, compared with WT mice, Sptlc1(+/-) and Sptlc2(+/-) mice had: (1) decreased liver Sptlc1 and Sptlc2 mRNA by 44% and 57% (P<0.01 and P<0.0001, respectively); (2) decreased liver Sptlc1 mass by 50% and Sptlc2 mass by 70% (P<0.01 and P<0.01, respectively), moreover, Sptlc1 mass decreased by 70% in Sptlc2(+/-) mouse liver, while Sptlc2 mass decreased by 53% in Sptlc1(+/-) mouse liver (P<0.001 and P<0.01, respectively); (3) decreased liver SPT activity by 45% and 60% (P<0.01, respectively); (4) decreased liver ceramide (22% and 39%, P<0.05 and P<0.01, respectively) and sphingosine levels (22% and 31%, P<0.05 and P<0.01, respectively); (5) decreased plasma ceramide (45% and 39%, P<0.01, respectively), sphingosine-1-phosphate (31% and 32%, P<0.01, respectively) and sphingosine levels (22.5% and 25%, P<0.01, respectively); (6) dramatically decreased plasma lysosphingomyelin (17-fold and 16-fold, P<0.0001, respectively); and (7) no change of plasma sphingomyelin, triglyceride, total cholesterol, phospholipids, and liver sphingomyelin levels. These results indicated that both Sptlc1 and Sptlc2 interactions are necessary for SPT activity in vivo, and that SPT activity directly influences plasma sphingolipid levels. Furthermore, manipulation of SPT activity might well influence the

  7. 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.

  8. 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

  9. The Fusarium oxysporum gnt2, encoding a putative N-acetylglucosamine transferase, is involved in cell wall architecture and virulence.

    PubMed

    López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M Isabel G

    2013-01-01

    With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

  10. Glutathione S-transferases gene polymorphisms and risk of male idiopathic infertility: a systematic review and meta-analysis.

    PubMed

    Li, Xin; Pan, Jinhong; Liu, Qigui; Xiong, Enqing; Chen, Zhiwen; Zhou, Zhansong; Su, Yongping; Lu, Gensheng

    2013-03-01

    The Glutathione S-transferases (GSTs) polymorphisms have been implicated in susceptibility to male idiopathic infertility, but study results are still controversial. To investigate the genetic associations between GSTs polymorphisms and risk of male idiopathic infertility, a systematic review and meta-analysis were performed. Meta-analysis was performed by pooling odds ratio (OR) with its corresponding 95 % confidence interval (95 % CI) form studies in electronic databases up to March 16, 2012. Glutathione S-transferase M 1 (GSTM1) null genotype, Glutathione S-transferase T 1 (GSTT1) null genotype, and dual null genotype of GSTM1/GSTT1 were analyzed independently. 14 eligible studies with a total of 1,845 idiopathic infertility males and 1,729 controls were included. There were 13 studies on GSTM1 polymorphism, 10 ones on GSTT1 polymorphism and 5 ones on GSTM1-GSTT1 interaction analysis. Meta-analyses of total relevant studies showed GSTM1 null genotype was significantly associated with an increased risk of male idiopathic infertility (OR = 1.40, 95 % CI 1.07-1.84, P OR = 0.015). The GSTM1-GSTT1 interaction analysis showed dual null genotype of GSTM1/GSTT1 was also significantly associated with increased risk of male idiopathic infertility (OR = 1.85, 95 % CI 1.07-3.21, P OR = 0.028). Subgroup analyses by ethnicity showed the associations above were still statistically significant in Caucasians (For GSTM1, OR = 1.51, 95 % CI 1.11-2.05, P OR = 0.009; For GSTM1/GSTT1, OR = 2.10, 95 % CI 1.51-2.91, P OR < 0.001). This meta-analysis suggests GSTM1 null genotype contributes to increased risk of male idiopathic infertility in Caucasians, and males with dual null genotype of GSTM1/GSTT1 are particularly susceptible to developing idiopathic infertility.

  11. Comparative study on glutathione transferases of rat brain and testis under the stress of phenobarbitol and β-methylcholanthrene*

    PubMed Central

    Thyagaraju, K.; Hemavathi, B.; Vasundhara, K.; Rao, A.D.; Devi, K.N.

    2005-01-01

    A comparative study was made on the tissue specific expression of glutathione transferases (GST) in brain and testis after exposure of rat to phenobarbitol (PB) and β-methylcholanthrene (MC). Glutathione transferases, a family of multifunctional proteins are involved in intracellular transport processes and in detoxication of electrophilic xenobiotics by catalyzing reactions such as conjugation, isomerization, reduction and thiolysis. On purification, the yield of GST proteins by affinity chromatography was 39% in testis and 32% in brain. The affinity purified testis GSTs were resolved by chromatofocusing into six anionic and four cationic isozymes, and in brain glutathione transferases were resolved into four anionic and three cationic isozymes, suggesting the presence of multiple isozymes with Yc, Yb, Yβ and Yδ in both of them. In testis and brain, these isozymes at identical pI values showed variable functions with a battery of substrates and the cationic isozymes of brain and testis showed identical properties in CHP (cumene hydroperoxide) at pH values of above 7.0. Substrate specificity studies and immunoblot analysis of testis and brain proteins revealed that they play a predominant role in the detoxication of phenobarbitol or β-methylcholanthrene. Expression of the isozymes in testis and brain on exposure to PB and MC indicated elevated subunit variation. In both testis and brain, Yδ of π class was expressed on PB treatment and Yc of α class and Yβ of μ class was expressed in MC treated testis and only Yc was predominantly expressed in MC treated brain. Thus these subunits expression is considered as markers for carcinogenesis and specific to chemical toxicity under phenobarbitol and β-methylcholanthrene stress. PMID:16052709

  12. 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.

  13. 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.

  14. Elucidation of catalytic specificities of human cytochrome P450 and glutathione S-transferase enzymes and relevance to molecular epidemiology.

    PubMed

    Guengerich, F P; Shimada, T; Raney, K D; Yun, C H; Meyer, D J; Ketterer, B; Harris, T M; Groopman, J D; Kadlubar, F F

    1992-11-01

    A number of different approaches have been used to determine the catalytic selectivity of individual human enzymes toward procarcinogens. Studies with cytochrome P450 (P450) enzymes and glutathione S-transferases are summarized here, and recent work with pyrrolizidine alkaloids, aflatoxins, 4,4'-methylenebis(2-chloroaniline), and ethyl carbamate is discussed. In some cases a single enzyme can catalyze both activation and detoxication reactions of a chemical. The purification and characterization of human lung P4501A1 and the development of a noninvasive assay for human P4502E1 are also described. PMID:1486866

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA- alkyl-transferase by protein engineering.

    PubMed

    Vettone, Antonella; Serpe, Mario; Hidalgo, Aurelio; Berenguer, José; del Monaco, Giovanni; Valenti, Anna; Rossi, Mosé; Ciaramella, Maria; Perugino, Giuseppe

    2016-01-01

    In the last decade, a powerful biotechnological tool for the in vivo and in vitro specific labeling of proteins (SNAP-tag™ technology) was proposed as a valid alternative to classical protein-tags (green fluorescent proteins, GFPs). This was made possible by the discovery of the irreversible reaction of the human alkylguanine-DNA-alkyl-transferase (hAGT) in the presence of benzyl-guanine derivatives. However, the mild reaction conditions and the general instability of the mesophilic SNAP-tag™ make this new approach not fully applicable to (hyper-)thermophilic and, in general, extremophilic organisms. Here, we introduce an engineered variant of the thermostable alkylguanine-DNA-alkyl-transferase from the Archaea Sulfolobus solfataricus (SsOGT-H5), which displays a catalytic efficiency comparable to the SNAP-tag™ protein, but showing high intrinsic stability typical of proteins from this organism. The successful heterologous expression obtained in a thermophilic model organism makes SsOGT-H5 a valid candidate as protein-tag for organisms living in extreme environments.

  1. Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

    SciTech Connect

    Garcia, Wanius; Travensolo, Regiane F.; Rodrigues, Nathalia C.; Muniz, João R. C.; Caruso, Célia S.; Lemos, Eliana G. M.; Araujo, Ana Paula U.; Carrilho, Emanuel

    2008-02-01

    Glutathione S-transferase from X. fastidiosa (xfGST) has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.23 Å. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 Å, α = 63.45, β = 80.66, γ = 94.55°. xfGST crystals diffracted to 2.23 Å resolution on a rotating-anode X-ray source.

  2. A cytosolic glutathione s-transferase, GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties.

    PubMed

    Arockiaraj, Jesu; Gnanam, Annie J; Palanisamy, Rajesh; Bhatt, Prasanth; Kumaresan, Venkatesh; Chaurasia, Mukesh Kumar; Pasupuleti, Mukesh; Ramaswamy, Harikrishnan; Arasu, Abirami; Sathyamoorthi, Akila

    2014-08-10

    Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P<0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.

  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. 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.

  5. Glutathione transferase theta in apical ciliary tuft regulates mechanical reception and swimming behavior of Sea Urchin Embryos

    PubMed Central

    Jin, Yinhua; Yaguchi, Shunsuke; Shiba, Kogiku; Yamada, Lixy; Yaguchi, Junko; Shibata, Daisuke; Sawada, Hitoshi; Inaba, Kazuo

    2013-01-01

    An apical tuft, which is observed in a wide range of embryos/larvae of marine invertebrates, is composed of a group of cilia that are longer and less motile than the abundant lateral cilia covering the rest of the embryonic surface. Although the apical tuft has been thought to function as a sensory organ, its molecular composition and roles are poorly understood. Here, we identified a glutathione transferase theta (GSTT) as an abundant and specific component of the apical tuft in sea urchin embryos. The expression of GSTT mRNA increases and becomes limited to the animal plate of the mesenchyme blastula, gastrula, and prism larva. Electron microscopy and tandem mass spectrometry demonstrated that the apical tuft contains almost every axonemal component for ciliary motility. Low concentrations of an inhibitor of glutathione transferase bromosulphophthalein (BSP) induce bending of apical tuft, suggesting that GSTT regulates motility of apical tuft cilia. Embryos treated with BSP swim with normal velocity and trajectories but show less efficiency of changing direction when they collide with an object. These results suggest that GSTT in the apical tuft plays an important role in the mechanical reception for the motility regulation of lateral motile cilia in sea urchin embryos. PMID:23907936

  6. Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer.

    PubMed

    Pucci, Sabina; Zonetti, Maria Josè; Fisco, Tommaso; Polidoro, Chiara; Bocchinfuso, Gianfranco; Palleschi, Antonio; Novelli, Giuseppe; Spagnoli, Luigi G; Mazzarelli, Paola

    2016-04-12

    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.

  7. Lipoprotein N-acyl transferase (Lnt1) is dispensable for protein O-mannosylation by Streptomyces coelicolor.

    PubMed

    Córdova-Dávalos, Laura Elena; Espitia, Clara; González-Cerón, Gabriela; Arreguín-Espinosa, Roberto; Soberón-Chávez, Gloria; Servín-González, Luis

    2014-01-01

    A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M. tuberculosis, Lnt1 of S. coelicolor does not interact with Ppm. The D2 catalytic domain of M. tuberculosisPpm was sufficient for complementation of an S. coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M. tuberculosisPmt was not active in S. coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.

  8. AN9, a Petunia Glutathione S-Transferase Required for Anthocyanin Sequestration, Is a Flavonoid-Binding Protein1

    PubMed Central

    Mueller, Lukas A.; Goodman, Christopher D.; Silady, Rebecca A.; Walbot, Virginia

    2000-01-01

    AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein. PMID:10938372

  9. 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

  10. 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

  11. 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

  12. Expression Patterns of Glutathione Transferase Gene (GstI) in Maize Seedlings Under Juglone-Induced Oxidative Stress

    PubMed Central

    Sytykiewicz, Hubert

    2011-01-01

    Juglone (5-hydroxy-1,4-naphthoquinone) has been identified in organs of many plant species within Juglandaceae family. This secondary metabolite is considered as a highly bioactive substance that functions as direct oxidant stimulating the production of reactive oxygen species (ROS) in acceptor plants. Glutathione transferases (GSTs, E.C.2.5.1.18) represent an important group of cytoprotective enzymes participating in detoxification of xenobiotics and limiting oxidative damages of cellular macromolecules. The purpose of this study was to investigate the impact of tested allelochemical on growth and development of maize (Zea mays L.) seedlings. Furthermore, the effect of juglone-induced oxidative stress on glutathione transferase (GstI) gene expression patterns in maize seedlings was recorded. It was revealed that 4-day juglone treatment significantly stimulated the transcriptional activity of GstI in maize seedlings compared to control plants. By contrast, at the 6th and 8th day of experiments the expression gene responses were slightly lower as compared with non-stressed seedlings. Additionally, the specific gene expression profiles, as well as the inhibition of primary roots and coleoptile elongation were proportional to juglone concentrations. In conclusion, the results provide strong molecular evidence that allelopathic influence of juglone on growth and development of maize seedlings may be relevant with an induction of oxidative stress in acceptor plants. PMID:22174645

  13. Post-transcriptional modification of the poly(A) length of galactose-1-phosphate uridyl transferase mRNA in Saccharomyces cerevisiae.

    PubMed Central

    Saunders, C A; Bostian, K A; Halvorson, H O

    1980-01-01

    Thermal elution poly(U)-Sepharose chromatography was utilized to fractionate yeast mRNA based on poly(A) size. Analysis of the in vitro translation products of the fractionated RNAs in a wheat-embryo cell-free protein synthesis system shows a heterogeneous but equal distribution of these abundant translatable mRNAs in the different poly(A) size classes. By comparing the translational activity of inducible galactose-1-phosphate uridyl transferase mRNA, which can be monitored as a function of age, to contitutive mRNAs, we demonstrate that initially galactose-1-phosphate uridyl transferase mRNA has a uniformly large poly(A) tail which becomes heterogeneous and shorter with age in the cytoplasm. These observations are consistent with the previously observed cytoplasmic poly(A) catabolism in yeast and with cytoplasmic post-transcriptional modification of the poly(A) length of galactose-1-phosphate uridyl transferase mRNA. Images PMID:6255420

  14. 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

    2015-10-20

    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.

  15. 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

  16. 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.

  17. Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1.

    PubMed

    Martos-Maldonado, Manuel C; Quesada-Soriano, Indalecio; García-Maroto, Federico; Vargas-Berenguel, Antonio; García-Fuentes, Luís

    2012-12-01

    The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene-glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1.

  18. Increase of gluthatione S-transferase, carboxyl esterase and carbonyl reductase in Fasciola hepatica recovered from triclabendazole treated sheep.

    PubMed

    Scarcella, S; Solana, M V; Fernandez, V; Lamenza, P; Ceballos, L; Solana, H

    2013-10-01

    Fasciolasis is a zoonotic parasitic disease caused by Fasciola hepatica and its control is mainly based on the use of triclabendazole (TCBZ). Parasite resistance to different anthelmintics is growing worldwide, including the resistance of F. hepatica to TCBZ. In the present work we evaluate "in vivo" the activity of xenobiotic metabolizing enzymes of phase I (carboxyl esterases) and phase II (glutathione S-transferases and carbonyl reductases) recovered of flukes from sheep treated with TCBZ. All three enzymes showed increased activity in TCBZ flukes returning 60h post-treatment at similar to baseline unexposed flukes. TCBZ action may induce secondary oxidative stress, which may explain the observed increment in activities of the analyzed enzymes as a defensive mechanism. The enzymes analyzed are candidates to participate actively in the development of resistance at TCBZ in F. hepatica.

  19. Transgenic Arabidopsis Plants Expressing Tomato Glutathione S-Transferase Showed Enhanced Resistance to Salt and Drought Stress

    PubMed Central

    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

  20. 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.

  1. Identification of the nuclear localisation signal of O-GlcNAc transferase and its nuclear import regulation

    PubMed Central

    Seo, Hyeon Gyu; Kim, Han Byeol; Kang, Min Jueng; Ryum, Joo Hwan; Yi, Eugene C.; Cho, Jin Won

    2016-01-01

    Nucleocytoplasmic O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues. Although the cellular localisation of OGT is important to regulate a variety of cellular processes, the molecular mechanisms regulating the nuclear localisation of OGT is unclear. Here, we characterised three amino acids (DFP; residues 451–453) as the nuclear localisation signal of OGT and demonstrated that this motif mediated the nuclear import of non-diffusible β-galactosidase. OGT bound the importin α5 protein, and this association was abolished when the DFP motif of OGT was mutated or deleted. We also revealed that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT. Our findings may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously. PMID:27713473

  2. DddD is a CoA-transferase/lyase producing dimethyl sulfide in the marine environment.

    PubMed

    Alcolombri, Uria; Laurino, Paola; Lara-Astiaso, Pedro; Vardi, Assaf; Tawfik, Dan S

    2014-09-01

    Dimethyl sulfide (DMS) is produced in oceans in vast amounts (>10(7) tons/year) and mediates a wide range of processes from regulating marine life forms to cloud formation. Nonetheless, none of the enzymes that produce DMS from dimethylsulfoniopropionate (DMSP) has been adequately characterized. We describe the expression and purification of DddD from the marine bacterium Marinomonas sp. MWYL1 and its biochemical characterization. We identified DMSP and acetyl-coenzyme A to be DddD's native substrates and Asp602 as the active site residue mediating the CoA-transferase prior to lyase activity. These findings shed light on the biochemical utilization of DMSP in the marine environment.

  3. 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.

  4. 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.

  5. Micro-plasticity of genomes as illustrated by the evolution of glutathione transferases in 12 Drosophila species.

    PubMed

    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.

  6. 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

  7. 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

  8. Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro.

    PubMed

    Klaus, Anna; Zorman, Sarah; Berthier, Alexandre; Polge, Cécile; Ramirez, Sacnicte; Michelland, Sylvie; Sève, Michel; Vertommen, Didier; Rider, Mark; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe

    2013-01-01

    AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

  9. 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

  10. A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency.

    PubMed

    Shafqat, Naeem; Kavanagh, Kate L; Sass, Jörn Oliver; Christensen, Ernst; Fukao, Toshiyuki; Lee, Wen Hwa; Oppermann, Udo; Yue, Wyatt W

    2013-11-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT . PMID:23420214

  11. 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

  12. 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.

  13. Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus.

    PubMed

    Patel, Kinnari B; Toh, Evelyn; Fernandez, Ximena B; Hanuszkiewicz, Anna; Hardy, Gail G; Brun, Yves V; Bernards, Mark A; Valvano, Miguel A

    2012-05-01

    Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.

  14. 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

  15. 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

  16. Association between glutathione S-transferase M1 null genotype and risk of gallbladder cancer: a meta-analysis.

    PubMed

    Sun, Hong-Li; Han, Bing; Zhai, Hong-Peng; Cheng, Xin-Hua; Ma, Kai

    2014-01-01

    Glutathione S-transferases (GSTs) are a family of enzymes which are involved in the detoxification of potential carcinogens. Glutathione S-transferase M1 (GSTM1) null genotype can impair the enzyme activity of GSTs and is suspected to increase the susceptibility to gallbladder cancer. Previous studies investigating the association between GSTM1 null genotype and risk of gallbladder cancer reported inconsistent findings. To quantify the association between GSTM1 null genotype and risk of gallbladder cancer, we performed a meta-analysis of published studies. We searched PubMed, Embase, and Wanfang databases for all possible studies. We estimated the pooled odds ratio (OR) with its 95% confidence interval (95% CI) to assess the association. Meta-analysis of total included studies showed that GSTM1 null genotype was not associated with gallbladder cancer risk (OR = 1.13, 95% CI 0.88-1.46, P = 0.332). Subgroup analysis by ethnicity showed that there was no association between GSTM1 null genotype and risk of gallbladder cancer in both Caucasians and Asians. However, meta-analysis of studies with adjusted estimations showed that GSTM1 null genotype was associated with increased risk of gallbladder cancer (OR = 1.46, 95% CI 1.02-2.09, P = 0.038). Thus, this meta-analysis shows that GSTM1 null genotype is likely to be associated with risk of gallbladder cancer. More studies with well design and large sample size are needed to further validate the association between GSTM1 null genotype and gallbladder cancer.

  17. 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

  18. Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

    PubMed Central

    Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

    2014-01-01

    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

  19. 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

  20. Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate production capacity in individuals according to diet and age.

    PubMed

    Hippe, Berit; Zwielehner, Jutta; Liszt, Kathrin; Lassl, Cornelia; Unger, Frank; Haslberger, Alexander G

    2011-03-01

    The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function.

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. Properties of Succinyl-Coenzyme A:l-Malate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus

    PubMed Central

    Friedmann, Silke; Steindorf, Astrid; Alber, Birgit E.; Fuchs, Georg

    2006-01-01

    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 (αβ)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

  6. Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium

    PubMed Central

    Ridlon, Jason M.; Hylemon, Phillip B.

    2012-01-01

    The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer. PMID:22021638

  7. 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

  8. 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

  9. Association of genetic polymorphism of glutathione S-transferase (GSTM1, GSTT1, GSTP1) with bladder cancer susceptibility.

    PubMed

    Safarinejad, Mohammad Reza; Safarinejad, Saba; Shafiei, Nayyer; Safarinejad, Shiva

    2013-10-01

    The glutathione-S-transferases (GSTs) comprise a class of enzymes that detoxify carcinogenic compounds by conjugating glutathione to facilitate their removal. Polymorphisms in GSTM1, GSTT1, and GSTP1 genes have been related to risk for bladder cancer. Studies focusing on GSTs gene variants relationship with the risk of bladder cancer have produced conflicting and inconsistent results. We examine the association between genetic polymorphism of glutathione S-transferase P1, GSTM1, GSTT1 genes and development of bladder transitional cell carcinoma (TCC). The study population consisted of 166 histologically confirmed male bladder TCC cases and 332 healthy male controls. Genotyping was done using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method and also investigated combined gene interactions. The GSTP1 Val/Val genotype was significantly associated with bladder cancer (OR = 4.32, 95% CI: 2.64-6.34), whereas the association observed for GSTM1 null (OR = 1.32, 95% CI: 0.82-2.62; P = 0.67) and GSTT1 null genotype (OR = 1.18, 95% CI: 0.79-1.67; P = 0.74) did not reach statistical significance. There was a significant multiple interaction between GSTM1, GSTT1, and GSTP1 genotypes in risk of bladder cancer (P for interaction = 0.02). The risk associated with the concurrent presence of GSTM1 positive and GSTP1 Ile/Val or Val/Val (OR = 3.71, 95% CI: 2.34-5.54) and GSTT1 positive and GSTP1 Ile/Val or Val/Val (OR = 2.66, 95% CI: 1.54-4.72) was statistically significant. Patients carrying GSTP1 Val/Val genotype were at increased risk for developing high-grade (OR = 7.68, 95% CI: 4.73-19.25) and muscle invasive (OR = 10.67, 95% CI: 6.34-21.75) bladder cancer. High risk for bladder TCC also was observed with respect to combined GSTT1 null/GSTP1 Ile/Val or Val/Val (OR = 4.76, 95% CI: 2.68-18.72) and GSTM1 null/GSTT1 null/GSTP1 Ile/Val or Val/Val (OR = 6.42, 95% CI: 4.76-14.72) genotype variant. This study suggests that the GSTP1 polymorphism

  10. Comparison of epsilon- and delta-class glutathione S-transferases: the crystal structures of the glutathione S-transferases DmGSTE6 and DmGSTE7 from Drosophila melanogaster.

    PubMed

    Scian, Michele; Le Trong, Isolde; Mazari, Aslam M A; Mannervik, Bengt; Atkins, William M; Stenkamp, Ronald E

    2015-10-01

    Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications. In addition, it is essential to fully understand the structure and function of invertebrate GSTs, which play important roles in basic biological processes. Invertebrates harbor delta- and epsilon-class GSTs, which are not found in other organisms. Drosophila melanogaster GSTs (DmGSTs) are likely to contribute to detoxication or antioxidative stress during development, but they have not been fully characterized. Here, the structures of two epsilon-class GSTs from Drosophila, DmGSTE6 and DmGSTE7, are reported at 2.1 and 1.5 Å resolution, respectively, and are compared with other GSTs to identify structural features that might correlate with their biological functions. The structures of DmGSTE6 and DmGSTE7 are remarkably similar; the structures do not reveal obvious sources of the minor functional differences that have been observed. The main structural difference between the epsilon- and delta-class GSTs is the longer helix (A8) at the C-termini of the epsilon-class enzymes.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Subcellular Golgi localization of stathmin family proteins is promoted by a specific set of DHHC palmitoyl transferases.

    PubMed

    Levy, Aurore D; Devignot, Véronique; Fukata, Yuko; Fukata, Masaki; Sobel, André; Chauvin, Stéphanie

    2011-06-01

    Protein palmitoylation is a reversible lipid modification that plays critical roles in protein sorting and targeting to specific cellular compartments. The neuronal microtubule-regulatory phosphoproteins of the stathmin family (SCG10/stathmin 2, SCLIP/stathmin 3, and RB3/stathmin 4) are peripheral proteins that fulfill specific and complementary roles in the formation and maturation of the nervous system. All neuronal stathmins are localized at the Golgi complex and at vesicles along axons and dendrites. Their membrane anchoring results from palmitoylation of two close cysteine residues present within their homologous N-terminal targeting domains. By preventing palmitoylation with 2-bromopalmitate or disrupting the integrity of the Golgi with brefeldin A, we were able to show that palmitoylation of stathmins 2 and 3 likely occurs at the Golgi and is crucial for their specific subcellular localization and trafficking. In addition, this membrane binding is promoted by a specific set of palmitoyl transferases that localize with stathmins 2 and 3 at the Golgi, directly interact with them, and enhance their membrane association. The subcellular membrane-associated microtubule-regulatory activity of stathmins might then be fine-tuned by extracellular stimuli controlling their reversible palmitoylation, which can be viewed as a crucial regulatory process for specific and local functions of stathmins in neurons.

  19. 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

  20. 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.

  1. Crystal Structure of Saccharomyces cerevisiae ECM4, a Xi-Class Glutathione Transferase that Reacts with Glutathionyl-(hydro)quinones

    PubMed Central

    Schwartz, Mathieu; Didierjean, Claude; Hecker, Arnaud; Girardet, Jean-Michel; Morel-Rouhier, Mélanie; Gelhaye, Eric; Favier, Frédérique

    2016-01-01

    Glutathionyl-hydroquinone reductases (GHRs) belong to the recently characterized Xi-class of glutathione transferases (GSTXs) according to unique structural properties and are present in all but animal kingdoms. The GHR ScECM4 from the yeast Saccharomyces cerevisiae has been studied since 1997 when it was found to be potentially involved in cell-wall biosynthesis. Up to now and in spite of biological studies made on this enzyme, its physiological role remains challenging. The work here reports its crystallographic study. In addition to exhibiting the general GSTX structural features, ScECM4 shows extensions including a huge loop which contributes to the quaternary assembly. These structural extensions are probably specific to Saccharomycetaceae. Soaking of ScECM4 crystals with GS-menadione results in a structure where glutathione forms a mixed disulfide bond with the cysteine 46. Solution studies confirm that ScECM4 has reductase activity for GS-menadione in presence of glutathione. Moreover, the high resolution structures allowed us to propose new roles of conserved residues of the active site to assist the cysteine 46 during the catalytic act. PMID:27736955

  2. Flock house virus RNA polymerase initiates RNA synthesis de novo and possesses a terminal nucleotidyl transferase activity.

    PubMed

    Wu, Wenzhe; Wang, Zhaowei; Xia, Hongjie; Liu, Yongxiang; Qiu, Yang; Liu, Yujie; Hu, Yuanyang; Zhou, Xi

    2014-01-01

    Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3'-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3' initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication. PMID:24466277

  3. 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.; 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

  4. 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.

  5. Expression of human glutathione S-transferases in Saccharomyces cerevisiae confers resistance to the anticancer drugs adriamycin and chlorambucil.

    PubMed Central

    Black, S M; Beggs, J D; Hayes, J D; Bartoszek, A; Muramatsu, M; Sakai, M; Wolf, C R

    1990-01-01

    Adaptation and resistance to chemicals in the environment is a critical part of the evolutionary process. As a result, a wide variety of defence systems that protect cells against chemical insult have evolved. Such chemical resistance mechanisms appear to play a central role in determining the sensitivity of human tumours to treatment with chemotherapeutic drugs. The glutathione S-transferases (GST) are important detoxification enzymes whose over-expression has been associated with drug-resistance. In order to evaluate this possibility we have expressed the human Alpha-class and Pi-class GST cDNAs that encode GST B1B1 and GST pi in the yeast Saccharomyces cerevisiae. The expression of GST B1B1 or GST pi resulted in a marked reduction in the cytotoxic effects of chlorambucil, a bifunctional alkylating agent, and an anthracycline, adriamycin. These data provide direct evidence that the over-expression of GST in cells can confer resistance to anticancer drugs. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:2194447

  6. 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

  7. 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

  8. 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

  9. 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.

  10. A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase

    PubMed Central

    1987-01-01

    Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition. PMID:3571328

  11. 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

  12. 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.

  13. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation.

    PubMed

    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

    2015-05-05

    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.

  14. 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.

  15. 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

  16. 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

  17. Characterization of a lambda-cyhalothrin metabolizing glutathione S-transferase CpGSTd1 from Cydia pomonella (L.).

    PubMed

    Liu, Jiyuan; Yang, Xueqing; Zhang, Yalin

    2014-11-01

    In insects, glutathione S-transferases (GSTs) are enzymes involved in detoxification of insecticides. However, few data are available for the codling moth, Cydia pomonella (L.). In this study, we cloned a delta class GST gene CpGSTd1 from C. pomonella. Real-time quantitative PCR shows that CpGSTd1 was up-regulated with aging, and the mRNA level of CpGSTd1 was higher in the fat body and silk glands than in other tissues. The expression level of CpGSTd1 exposure to insecticide suggests that CpGSTd1 is up-regulated after chlorpyrifos-methyl and lambda-cyhalothrin treatments. Both lambda-cyhalothrin and chlorpyrifos-methyl altered GST activity in vivo. The purified CpGSTd1 protein exhibits a high catalytic efficiency with CDNB and was inhibited by lambda-cyhalothrin and chlorpyrifos-methyl in vitro. Metabolism assays indicate that lambda-cyhalothrin was significantly metabolized while chlorpyrifos-methyl was not metabolized by CpGSTd1. Binding free energy analysis suggests that CpGSTd1 binding is tighter with lambda-cyhalothrin than with chlorpyrifos-methyl. Our study suggests that CpGSTd1 plays a key role in the metabolism of insecticides in C. pomonella. PMID:24827500

  18. Role of the 4-phosphopantetheinyl transferase of Trichoderma virens in secondary metabolism and induction of plant defense responses.

    PubMed

    Velazquez-Robledo, R; Contreras-Cornejo, H A; Macias-Rodriguez, L; Hernandez-Morales, A; Aguirre, J; Casas-Flores, S; Lopez-Bucio, J; Herrera-Estrella, A

    2011-12-01

    Trichoderma virens is a ubiquitous soil fungus successfully used in biological control due to its efficient colonization of plant roots. In fungi, 4-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary and secondary metabolism. Therefore, we cloned the PPTase gene ppt1 from T. virens and generated PPTase-deficient (?ppt1) and overexpressing strains to investigate the role of this enzyme in biocontrol and induction of plant defense responses. The ?ppt1 mutants were auxotrophic for lysine, produced nonpigmented conidia, and were unable to synthesize nonribosomal peptides. Although spore germination was severely compromised under both low and high iron availability, mycelial growth occurred faster than the wild type, and the mutants were able to efficiently colonize plant roots. The ?ppt1 mutants were unable of inhibiting growth of phytopathogenic fungi in vitro. Arabidopsis thaliana seedlings co-cultivated with wild-type T. virens showed increased expression of pPr1a:uidA and pLox2:uidA markers, which correlated with enhanced accumulation of salicylic acid (SA), jasmonic acid, camalexin, and resistance to Botrytis cinerea. Co-cultivation of A. thaliana seedlings with ?ppt1 mutants compromised the SA and camalexin responses, resulting in decreased protection against the pathogen. Our data reveal an important role of T. virens PPT1 in antibiosis and induction of SA and camalexin-dependent plant defense responses.

  19. 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.

  20. Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos.

    PubMed

    Levy, R; Benchaib, M; Cordonier, H; Souchier, C; Guerin, J F

    1998-08-01

    Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilization. Observation of the cellular actin cortex and chromatin showed a high frequency of embryos with blastomeres exhibiting two or more nuclei, while others had nuclei displaying chromatin condensation and fragmentation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigated using two detection methods for cells undergoing apoptosis. Detection of phosphatidylserine exposure was performed using annexin V; the chromosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which continued to develop normally after thawing. The TUNEL assay was positive in 30% (15/50) of arrested embryos, all of which had cytoplasmic fragments. In contrast, embryos showing regular size blastomeres without fragments were TUNEL negative.

  1. Genome-Wide Analysis of the Glutathione S-Transferase Gene Family in Capsella rubella: Identification, Expression, and Biochemical Functions.

    PubMed

    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. PMID:27630652

  2. 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

  3. 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

  4. 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.

  5. Arabidopsis RAB geranylgeranyl transferase beta-subunit mutant is constitutively photomorphogenic, and has shoot growth and gravitropic defects.

    PubMed

    Hála, Michal; Soukupová, Hana; Synek, Lukás; Zárský, Viktor

    2010-05-01

    RAB GTPases are important directional regulators of intracellular vesicle transport. Membrane localization of RAB GTPases is mediated by C-terminal double geranylgeranylation. This post-translational modification is catalyzed by the alpha-beta-heterodimer catalytic core of RAB geranylgeranyl transferase (RAB-GGT), which cooperates with the RAB escort protein (REP) that presents a nascent RAB. Here, we show that RAB-geranylgeranylation activity is significantly reduced in two homozygous mutants of the major Arabidopsis beta-subunit of RAB-GGT (AtRGTB1), resulting in unprenylated RAB GTPases accumulation in the cytoplasm. Both endocytosis and exocytosis are downregulated in rgtb1 homozygotes defective in shoot growth and morphogenesis. Root gravitropism is normal in rgtb1 roots, but is significantly compromised in shoots. Mutants are defective in etiolation and show constitutive photomorphogenic phenotypes that cannot be rescued by brassinosteroid treatment, similarly to the det3 mutant that is also defective in the secretory pathway. Transcriptomic analysis revealed an upregulation of specific RAB GTPases in etiolated wild-type plants. Taken together, these data suggest that the downregulation of the secretory pathway is interpreted as a photomorphogenic signal in Arabidopsis.

  6. Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif.

    PubMed

    Yalovsky, S.; Loraine, A. E.; Gruissem, W.

    1996-04-01

    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.

  7. 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.

  8. 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

  9. Evaluation of the non-catalytic binding function of Ts26GST a glutathione transferase isoform of Taenia solium.

    PubMed

    Plancarte, A; Romero, J R; Nava, G; Reyes, H; Hernández, M

    2014-03-01

    Taenia solium glutathione transferase isoform of 26.5 kDa (Ts26GST) was observed to bind non-catalytically to porphyrins, trans-trans-dienals, bile acids and fatty acids, as assessed by inhibition kinetics, fluorescence spectroscopy and competitive fluorescence assays with 8-anilino-1-naphthalene sulfonate (ANS). The quenching of Ts26GST intrinsic fluorescence allowed for the determination of the dissociation constants (KD) for all ligands. Obtained data indicate that Ts26GST binds to all ligands but with different affinity. Porphyrins and lipid peroxide products inhibited Ts26GST catalytic activity up to 100% in contrast with only 20-30% inhibition observed for bile acids and two saturated fatty acids. Non-competitive type inhibition was observed for all enzyme inhibitor ligands except for trans-trans-2,4-decadienal, which exhibited uncompetitive type inhibition. The dissociation constant value KD = 0.7 μM for the hematin ligand, determined by competitive fluorescence assays with ANS, was in good agreement with its inhibition kinetic value Ki = 0.3 μM and its intrinsic fluorescence quenching KD = 0.7 μM. The remaining ligands did not displace ANS from the enzyme suggesting the existence of different binding sites. In addition to the catalytic activity of Ts26GST the results obtained suggest that the enzyme exhibits a ligandin function with broad specificity towards nonsubstrate ligands.

  10. Antioxidant defense markers modulated by glutathione S-transferase genetic polymorphism: results of lung cancer case–control study

    PubMed Central

    Reszka, Edyta; Gromadzinska, Jolanta

    2007-01-01

    Oxidative stress and xenobiotic metabolizing enzymes are suspected to be related to carcinogenesis by different cellular mechanisms. Hence, our study aimed at identifying potential relationships between antioxidant defense parameters measured in blood and glutathione S-transferase (GST) genetic polymorphisms of four GST izoenzymes in lung cancer patients and reference individuals. The case–control study included 404 lung cancer patients and 410 non-cancer subjects as controls, matched by age, gender and place of living (central Poland). In control subjects with GSTM3*A/*A, GSTT1 null, GSTM1 null + GSTT1 null, GSTM3*A/*A + GSTT1 null genotype, glutathione peroxidase activity was significantly higher (P < 0.05) than in controls possessing respective potential protective GST genotypes. Controls with GSTM3*A/*A + GSTP1*B genotype presented significantly higher ceruloplasmin activity (P < 0.05) than GSTM3*B + GSTP1*A/*A carriers. Zinc level was significantly higher (P < 0.05) in controls and cases with GSTP1*B + GSTT1 null genotype and in cases with GSTM1 null + GSTP1*B genotype, when compared with respective potential protective GST genotypes. This case–control study indicates that particular defective GST genotypes may enhance the defense against oxidative stress. The potential relationship between the investigated antioxidative enzymes and microelements, and common functional genetic polymorphism of GST was observed mostly in control subjects. PMID:18850183

  11. Antioxidant defense markers modulated by glutathione S-transferase genetic polymorphism: results of lung cancer case-control study.

    PubMed

    Reszka, Edyta; Wasowicz, Wojciech; Gromadzinska, Jolanta

    2007-12-01

    Oxidative stress and xenobiotic metabolizing enzymes are suspected to be related to carcinogenesis by different cellular mechanisms. Hence, our study aimed at identifying potential relationships between antioxidant defense parameters measured in blood and glutathione S-transferase (GST) genetic polymorphisms of four GST izoenzymes in lung cancer patients and reference individuals. The case-control study included 404 lung cancer patients and 410 non-cancer subjects as controls, matched by age, gender and place of living (central Poland). In control subjects with GSTM3*A/*A, GSTT1 null, GSTM1 null + GSTT1 null, GSTM3*A/*A + GSTT1 null genotype, glutathione peroxidase activity was significantly higher (P < 0.05) than in controls possessing respective potential protective GST genotypes. Controls with GSTM3*A/*A + GSTP1*B genotype presented significantly higher ceruloplasmin activity (P < 0.05) than GSTM3*B + GSTP1*A/*A carriers. Zinc level was significantly higher (P < 0.05) in controls and cases with GSTP1*B + GSTT1 null genotype and in cases with GSTM1 null + GSTP1*B genotype, when compared with respective potential protective GST genotypes. This case-control study indicates that particular defective GST genotypes may enhance the defense against oxidative stress. The potential relationship between the investigated antioxidative enzymes and microelements, and common functional genetic polymorphism of GST was observed mostly in control subjects. PMID:18850183

  12. Evaluation of glutathione S-transferase genetic variants affecting type 2 diabetes susceptibility: a meta-analysis.

    PubMed

    Tang, Song-Tao; Wang, Chang-Jiang; Tang, Hai-Qin; Zhang, Qiu; Wang, Yuan

    2013-11-10

    Genetic polymorphisms of glutathione S-transferases (GSTs) and type 2 diabetes mellitus (T2DM) risk have been widely studied, however, the results were somewhat conflicting. To evaluate the association of GSTs (GSTM1, GSTT1 and GSTP1) gene polymorphisms with T2DM, a meta-analysis was performed before October, 2012. ORs were pooled according to random-effects model. There were a total of 1354/1666 (n=9) cases/controls (studies) for GSTM1, 1271/1470 (n=8) for GSTT1, and 1205/1250 (n=7) for GSTM1. There were significant associations between GSTM1 polymorphism, GSTT1 polymorphism and T2DM in the contrast of present genotype vs. null genotype, with pooled OR=1.99 (95%CI=1.46-2.71) and OR=1.61 (95%CI=1.19-2.17), respectively. Yet no significant association of GSTP1 polymorphism and T2DM was showed. When stratified by ethnicity, the significant associations were also existed in Asians for GSTM1 and GSTT1, but not GSTP1. No publication bias but some extent of heterogeneity was observed. Finally, the accumulated evidence proved the obvious associations of GSTM1 and GSTT1 polymorphisms with an increased risk of T2DM.

  13. PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

    PubMed

    Kim, Youseung; Maciag, Anna E; Cao, Zhao; Deschamps, Jeffrey R; Saavedra, Joseph E; Keefer, Larry K; Holland, Ryan J

    2015-08-01

    PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.

  14. Metabolism of cisplatin in the organs of Rattus norvegicus: role of Glutathione S-transferase P1.

    PubMed

    Nagar, Ritika; Khan, Amir Riyaz; Poonia, Anuj; Mishra, Pankaj Kishor; Singh, Simendra

    2015-03-01

    Glutathione S-transferases (GSTs) play an important role in the biotransformation of endogenous compounds and xenobiotics as well as in the metabolic inactivation of pharmacologically active substances, including anticancer drugs. Using cisplatin as the prototype drug, we investigated if any correlation exists between GSH levels, GSTs/GSTP1 activity and the fate of cisplatin in different organs of Rattus norvegicus. GSH-cisplatin complex was prepared, purified by anion-exchange chromatography and subjected to mass spectroscopic analysis which confirmed the structure to be diglutathione-monoplatinum (diglutathionylplatinum). Purified diglutathionylplatinum was used to quantify metabolite formed in different tissue homogenates. Specific GSTP1 activity was found to be highest in kidneys, which correlated positively with the levels of metabolite formed in renal tissues. Altogether, our results showed that cisplatin metabolism in different organs of rats correlated positively with specific GSTP1 activities and this enzyme may be a critical determinant of extent of cellular uptake or retention of cisplatin in renal and liver tissues.

  15. Glutathione-S-transferases M1/T1 gene polymorphisms and endometriosis: a meta-analysis in Chinese populations.

    PubMed

    Chen, Xin-Ping; Xu, Da-Feng; Xu, Wei-Hua; Yao, Jia; Fu, Sheng-Miao

    2015-01-01

    In view of the controversies surrounding the glutathione-S-transferases (GST) M1/T1-endometriosis association, a meta-analysis of the GSTM1/GSTT1 genetic association studies of endometriosis was performed in Chinese populations. PubMed, Springer Link, OvidSP, and Chinese databases were searched for related studies. A total of nine studies on GSTM1-endometriosis involved 874 cases and 997 controls, and five studies on GSTT1 involved 404 cases and 513 controls were included in this meta-analysis. Overall, the null genotype of GSTM1/GSTT1 was significantly related to endometriosis risk in Chinese populations (GSTM1, OR = 2.21, 95% CI: 1.22-4.01; GSTT1, OR = 2.31, 95% CI: 1.34-3.99). In subgroup analyses stratified by ethnicity and source of controls, the same results were observed in Chinese Han and population-based studies. The sensitivity analysis confirmed the reliability and stability of the meta-analysis. No publication bias was found among studies by Egger's test. In conclusion, our meta-analysis supports that the GSTM1/GSTT1 null genotype might contribute to individual susceptibility to endometriosis in Chinese populations, especially in Chinese Han.

  16. 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.

  17. 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.

  18. DNA damage and effects on glutathione-S-transferase activity induced by atrazine exposure in zebrafish (Danio rerio).

    PubMed

    Zhu, Lusheng; Dong, Xiaoli; Xie, Hui; Wang, Jun; Wang, Jinhua; Su, Jun; Yu, Changwei

    2011-10-01

    This study was undertaken to investigate the protective effect of atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine) on the activity of glutathione-S-transferase (GST) and DNA damage in males and females of adult zebrafish (Danio rerio). Zebrafish were exposed to control and three treatments (0.01, 0.1, and 1 mg/L) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, for males, the GST activity at lower atrazine concentrations (0.01 and 0.1 mg/L) was markedly higher than that of the controls throughout the duration of the experiment while there was a significant inhibition of the GST activity at 1 mg/L atrazine at days 5 and 20. For females, a significant increase was detected at 0.1 mg/L on the days 5 and 15 and at 0.01 mg/L on day 20. The DNA damage in zebrafish was evaluated using the comet assay; the olive tail moments obtained for hepatopancreas were enhanced after treatment with different concentrations of atrazine on days 5, 10, 15, 20, and 25. The DNA damage increased with increasing atrazine concentrations, indicating that genotoxicity of atrazine and significant differences was found compared to the controls. In conclusion, these findings provide further evidence of the effects of atrazine on aquatic ecosystems.

  19. 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

  20. 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.

  1. 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.

  2. Exploring the transferase activity of Ffase from Schwanniomyces occidentalis, a β-fructofuranosidase showing high fructosyl-acceptor promiscuity.

    PubMed

    Piedrabuena, David; Míguez, Noa; Poveda, Ana; Plou, Francisco J; Fernández-Lobato, María

    2016-10-01

    The β-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces the prebiotic sugars 6-kestose and 1-kestose by transfructosylation of sucrose, which makes it of biotechnological interest. In this study, the hydrolase and transferase activity of this enzyme was kinetically characterized and its potential to synthesize new fructosylated products explored. A total of 40 hydroxylated compounds were used as potential fructosyl-acceptor alternatives to sucrose. Only 17 of them, including some monosaccharides, disaccharides, and oligosaccharides as well as alditols and glycosides were fructosylated. The best alternative acceptors were the alditols. The major transfer product of the reaction including mannitol was purified and characterized as 1-O-β-D-fructofuranosyl-D-mannitol, whose maximum concentration reached 44 g/L, representing about 7.3 % of total compounds in the mixture and 89 % of all products generated by transfructosylation. The reactions including erythritol produced 35 g/L of an isomer mixture comprising 1- and 4-O-β-D-fructofuranosyl-D-erythritol. In addition, Ffase produced 24 g/L of the disaccharide blastose by direct fructosylation of glucose, which makes it the first enzyme characterized from yeast showing this ability. Thus, novel fructosylated compounds with potential applications in food and pharmaceutical industries can be obtained due to the Ffase fructosyl-acceptor promiscuity.

  3. Polymorphism of cytochrome p450, glutathione-s-transferase and N-acetyltransferases: influence on lung cancer susceptibility.

    PubMed

    Shukla, R K; Kant, S; Mittal, B; Bhattacharya, S

    2010-01-01

    Lung cancer remains a major health challenge in the world. It is the commonest cause of cancer mortality in men, it has been suggested that genetic susceptibility may contribute to the major risk factor, with increasing prevalence of smoking. Lung cancer has reached epidemic proportions in India. Recently indoor air pollution and dietary factors have been implicated in the causation of lung Cancer development. Accumulating evidences have highlighted that several polymorphisms involve the metabolic activation or detoxification of carcinogens derived from cigarette smoke have been found to be associated with lung cancer risk. Many studies have focused on the relation between the distribution of polymorphic variants of different forms of the metabolic enzymes and lung cancer susceptibility, Few of human biotransformating enzymes (Phase I enzyme: Cytochrome p450 enzymes, and Phase II enzymes: Glutathione-s-transferases, N-acetyltransferases) have been implicated in the formation and scavenging of ultimate reactive metabolites. These enzyme families are known to catalyze detoxification of electrophilic compounds including carcinogens. The treatment and prevention of lung cancer are major unmet needs that can probably be improved by a better understanding of the molecular origins and evolution of the disease. This review will focus on major recent advances in the molecular study of the origins and biology of lung cancer.

  4. 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

  5. Relationship between two tandemly arranged and light-induced glutathione S-transferase genes from the ciliated protozoa Blepharisma japonicum.

    PubMed

    Takada, Yuichi; Matsuoka, Tatsuomi

    2008-01-01

    Recently we reported a light-induced cDNA encoding glutathione S-transferase (GST) from the ciliated protozoa Blepharisma japonicum, which possessed photosensitive pigments. In this study, a novel cDNA encoding GST was further isolated, and the two GSTs (BjGST1 and BjGST2) showed high sequence identity of 86%. Phylogenetic trees indicated that the BjGSTs were distantly related to known classes of GSTs, and they could form a protozoa-specific class. The recombinant proteins also existed as homo- or heterodimers that exhibited different enzyme activities, appreciating the functional differentiation. Furthermore, the transcription levels of BjGST genes were coordinately regulated in response to light stimulation. In addition, the genomic structure analysis revealed that the two genes were tandemly arranged through an approximately 500-bp spacer region of unusual DNA structure containing cis-acting elements related to oxidative stress response. These results demonstrate that the two BjGSTs are expressed simultaneously and act cooperatively against photooxidative stress.

  6. 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. PMID:27630652

  7. Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens.

    PubMed

    Hoernstein, Sebastian N W; Mueller, Stefanie J; Fiedler, Kathrin; Schuelke, Marc; Vanselow, Jens T; Schuessele, Christian; Lang, Daniel; Nitschke, Roland; Igloi, Gabor L; Schlosser, Andreas; Reski, Ralf

    2016-06-01

    Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol. , DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (β-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.Data are available via ProteomeXchange with identifier PXD003228 and PXD003232.

  8. Cloning, characterisation and bacterial expression of full length cDNA for the mouse liver microsomal glutathione S-transferase.

    PubMed

    Raza, H; Mullick, J; John, A; Bhagwat, S V; Avadhani, N G

    2000-01-01

    We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.

  9. 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

  10. 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

  11. 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

  12. Neurokinin 3 receptor and phosphocholine transferase: missing factors for pathogenesis of C-reactive protein in preeclampsia

    PubMed Central

    Parchim, Nicholas F.; Wang, Wei; Iriyama, Takayuki; Ashimi, Olaide A.; Siddiqui, Athar H.; Blackwell, Sean; Sibai, Baha; Kellems, Rodney E.; Xia, Yang

    2015-01-01

    C-reactive protein (CRP), an innate immune mediator, is elevated in the circulation prior to symptoms in patients with preeclampsia (PE), a severe hypertensive pregnancy disorder with high mortality and morbidity. However, the specific sources underlying increased CRP and the role of elevated CRP in PE are undefined. Here, we report that circulating CRP levels are significantly increased in a large cohort of normotensive pregnant individuals compared to nulligravid women and is further increased in PE patients. These findings led us to further discover that placental syncytiotrophoblasts are previously unrecognized cellular sources of CRP and underlie elevated CRP in normotensive pregnant women and the additional increase in PE patients. Next, we demonstrated that injection of CRP induces PE features including hypertension (157.08 mmHg CRP treated vs. 118.99 mmHg control), proteinuria (35.0 mg/μg CRP treated vs. 14.1 mg/μg control), kidney and placental damage and increased levels of sFlt-1 in pregnant mice but not nonpregnant mice. We hypothesize that phosphocholine transferase, a placental specific enzyme posttranslationally modifying neurokinin B (NKB), is essential for the pathogenic role of CRP in PE through activation of the neurokinin 3 receptor. Overall, our studies have provided significant new insight regarding the pathogenic role of CRP in PE and highlighted innovative therapeutic strategies. PMID:25452470

  13. 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

  14. Catechol-O-methyl Transferase and Expression of Schizophrenia in 73 Adults with 22q11 Deletion Syndrome

    PubMed Central

    Bassett, Anne S.; Caluseriu, Oana; Weksberg, Rosanna; Young, Donald A.; Chow, Eva W.C.

    2011-01-01

    Background Catechol-O-methyl transferase (COMT) is a candidate gene for schizophrenia with a role in dopamine metabolism, particularly in frontal cortex. COMT is within the region commonly deleted in 22q11 deletion syndrome (22q11DS), a syndrome with high prevalence of schizophrenia. We examined the role of COMT in schizophrenia-related expression in 22q11DS. Methods We genotyped the COMT functional Val158/108Met allele in 73 Caucasian adults with 22q11DS (36 men, 37 women; aged 33.8, SD 10.1 years; 37 Met, 36 Val hemizygosity) blind to clinical data and assessed effects on symptoms and frontal functioning. Results The lower activity Met allele was not significantly more prevalent than the Val allele in 33 subjects with schizophrenia. Excitement symptoms were more severe, however, and three frontal cognitive tests (theory of mind, Trails B, and olfactory identification), communication, and social functioning measures showed significantly worse performance with Met allele hemizygosity, even after accounting for effects of schizophrenia. Conclusions The results suggest that hemizygosity of the COMT functional allele exerts an effect on some measures of frontal functioning in 22q11DS. Elevated levels of tonic dopamine activation associated with the COMT Met allele may underlie these aspects of expression. We must look elsewhere for causes of the high prevalence of schizophrenia in 22q11DS, however. PMID:17217925

  15. Glutathione S-transferase iso-enzymes in perfusate from pumped kidneys are associated with delayed graft function.

    PubMed

    Hall, I E; Bhangoo, R S; Reese, P P; Doshi, M D; Weng, F L; Hong, K; Lin, H; Han, G; Hasz, R D; Goldstein, M J; Schröppel, B; Parikh, C R

    2014-04-01

    Accurate and reliable assessment tools are needed in transplantation. The objective of this prospective, multi-center study was to determine the associations of the alpha and pi iso-enzymes of glutathione S-transferase (GST), measured from perfusate solution at the start and end (base and post) of kidney allograft machine perfusion, with subsequent delayed graft function (DGF). We also compared GST iso-enzyme perfusate levels from discarded versus transplanted kidneys. A total of 428 kidneys were linked to outcomes as recorded by the United Network of Organ Sharing. DGF, defined as any dialysis in the first week of transplant, occurred in 141 recipients (32%). Alpha- and pi-GST levels significantly increased during machine perfusion. The adjusted relative risks (95% confidence interval) of DGF with each log-unit increase in base and post pi-GST were 1.14 (1.0-1.3) and 1.36 (1.1-1.8), respectively. Alpha-GST was not independently associated with DGF. There were no significant differences in GST values between discarded and transplanted kidneys, though renal resistance was significantly higher in discarded kidneys. We found pi-GST at the end of machine perfusion to be independently associated with DGF. Further studies should elucidate the utility of GST for identifying injured kidneys with regard to organ allocation, discard and recipient management decisions.

  16. Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens.

    PubMed

    Hoernstein, Sebastian N W; Mueller, Stefanie J; Fiedler, Kathrin; Schuelke, Marc; Vanselow, Jens T; Schuessele, Christian; Lang, Daniel; Nitschke, Roland; Igloi, Gabor L; Schlosser, Andreas; Reski, Ralf

    2016-06-01

    Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol. , DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (β-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.Data are available via ProteomeXchange with identifier PXD003228 and PXD003232. PMID:27067052

  17. 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

  18. 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

  19. 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

  20. Immunogenicity of genetically engineered glutathione S-transferase fusion proteins containing a T-cell epitope from diphtheria toxin.

    PubMed Central

    Pillai, S; Dermody, K; Metcalf, B

    1995-01-01

    Glutathione S-transferase (GST) has been shown to induce a marginal antibody response in experimental animals as well as partial protection against a number of parasitic worms, including Schistosoma and Fasciola species. The objective of our study was to increase the immunogenicity of GST by adding heterologous T-cell epitopes at the carboxy terminus of the protein. We generated recombinant GST proteins by attaching one or three tandem repeats of a T-cell epitope of CRM197, a nontoxic variant of diphtheria toxin. This T-cell epitope encoding the region of amino acids 366 to 383 of CRM197, when contained in a GST fusion protein and/or after purification as a recombinant peptide, retained the ability to induce a CRM197-specific T-cell response. The fusion protein containing a single T-cell epitope induced a strong T-cell proliferative response to GST and also enhanced anti-GST antibody production in mice. The addition of three repeats of the epitope did not augment the responses when compared with the responses of GST itself. The results suggest that the addition of a single T-cell epitope to a larger protein like GST increases the immunogenicity of the protein. PMID:7534277

  1. Production of fructosyl transferase by Aspergillus oryzae CFR 202 in solid-state fermentation using agricultural by-products.

    PubMed

    Sangeetha, P T; Ramesh, M N; Prapulla, S G

    2004-10-01

    Fructosyl transferase (FTase) production by Aspergillus oryzae CFR 202 was carried out by solid-state fermentation (SSF), using various agricultural by-products like cereal bran, corn products, sugarcane bagasse,cassava bagasse (tippi) and by-products of coffee and tea processing. The FTase produced was used for the production of fructo-oligosaccharides (FOS), using 60% sucrose as substrate. Among the cereal bran used, rice bran and wheat bran were good substrates for FTase production by A. oryzae CFR 202. Among the various corn products used, corn germ supported maximum FTase production, whereas among the by-products of coffee and tea processing used, spent coffee and spent tea were good substrates, with supplementation of yeast extract and complete synthetic media. FTase had maximum activity at 60 degrees C and pH 6.0. FTase was stable up to 40 degrees C and in the pH range 5.0-7.0. Maximum FOS production was obtained with FTase after 8 h of reaction with 60% sucrose. FTase produced by SSF using wheat bran was purified 107-fold by ammonium sulphate precipitation (30-80%), DEAE cellulose chromatography and Sephadex G-200 chromatography. The molecular mass of the purified FTase was 116.3 kDa by SDS-PAGE. This study indicates the potential for the use of agricultural by-products for the efficient production of FTase enzyme by A. oryzae CFR 202 in SSF, thereby resulting in value addition of those by-products.

  2. Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti: a target for drug development against adult worm.

    PubMed

    Nathan, Sivaramakrishnan Thirumalai; Mathew, Nisha; Kalyanasundaram, Muthuswami; Balaraman, Kothandapani

    2005-06-01

    A three dimensional structural model of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. The three dimensional X-ray crystal structure of porcine pi-class GST with PDB ID: 2gsr-A chain protein with 42% sequential and functional homology was used as the template. The model of wbGST built by MODELLER6v2 was analyzed by the PROCHECK programs. Ramachandran plot analysis showed that 93.5% of the residues are in the core region followed by 5.4 and 1.1% residues in the allowed and generously allowed regions, respectively. None of the non-glycine residues is in disallowed regions. The PROSA II z-score and the energy graph for the final model further confirmed the quality of the modeled structure. The computationally modeled three-dimensional (3D) structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668). 1SFM was used for docking with GST inhibitors by Hex4.2 macromolecular docking using spherical polar Fourier correlations.

  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. 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

  5. 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

  6. Some effects of the fungicide propiconazole on cytochrome P450 and glutathione S-transferase in brown trout (Salmo trutta).

    PubMed

    Egaas, E; Sandvik, M; Fjeld, E; Källqvist, T; Goksøyr, A; Svensen, A

    1999-03-01

    The fungicide propiconazole (1-(2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl) -1H-1,2,4-triazole) induced the hepatic cytochrome P4501A (CYP1A) activity towards ethoxyresorufin-O-deethylase (EROD), the content of CYP1A protein as quantified by enzyme-linked immunosorbent assay (ELISA) and the glutathione S-transferase (GST) activity towards the three commonly used substrates CDNB(1-chloro-2,4-dinitrobenzene), cumene hydroperoxide (CU) and ethachrynic acid (EA) in brown trout (Salmo trutta) depending on dose and body weight. An exponential dose response relationship existed between propiconazole exposure and CYP1A activity. A 2. order polynomial regression of the propiconazole concentration (square root transformed) on the data for CDNB, EU and CU revealed a bell-shaped pattern of the GST induction. Reverse-phase HPLC of the GSH-affinity chromatography purified GST isozymes in trout exposed to respectively 8.3, 23, 93, 313 and 606 microg l(-1) propiconazole in the water indicated that the propiconazole treatment may lead to changes in the composition of the subunits compared to the controls. Thus, propiconazole exposure through the water changed the properties of the brown trout hepatic CYP1A and GST, and these changes may be used as a bioindicator on the molecular level of exposure and effect of propiconazole in controlled experiments. The use in monitoring of propiconazole exposure under natural field conditions is possible, however needs further investigation.

  7. Cloning of a novel glutathione S-transferase 3 (GST3) gene and expressionanalysis in pearl oyster, Pinctada martensii.

    PubMed

    Chen, Jinhui; Xiao, Shu; Deng, Yuewen; Du, Xiaodong; Yu, Ziniu

    2011-12-01

    Microsomal glutathione S-transferase (MGST) functions in cellular defense against xenobiotics and provides protection against the action of lipid hydroperoxides produced as a consequence of oxidative stress. In this study, a full-length cDNA encoding MGST3 (referred to as PmMGST3) was identified from the pearl oyster, Pinctada martensii by a combination of expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmMGST3 is 971 bp and contains a 5' UTR of 39 bp, a 3' UTR of 491 bp with a canonical polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) of 447 bp encoding a polypeptide of 146 residues. The deduced polypeptide contains a conserved motif (FNCx(1)QRx(2)H) characteristic of the MGST3 subfamily. The PmMGST3 transcript could be detected in all tissues tested, with highest transcript level seen in hepatopancreas. Cadmium treatment significantly increased PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depressed mRNA levels and then increased its level in haemocytes, gill and hepatopancreas in a time-dependent manner. In an assay using cumene hydroperoxide as a substrate, we demonstrated that PmMGST3 possesses glutathione-dependent peroxidase activity. These results suggest that PmMGST3 plays an important role in cellular defense against oxidative stress caused by cadmium and bacteria.

  8. Exploring the transferase activity of Ffase from Schwanniomyces occidentalis, a β-fructofuranosidase showing high fructosyl-acceptor promiscuity.

    PubMed

    Piedrabuena, David; Míguez, Noa; Poveda, Ana; Plou, Francisco J; Fernández-Lobato, María

    2016-10-01

    The β-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces the prebiotic sugars 6-kestose and 1-kestose by transfructosylation of sucrose, which makes it of biotechnological interest. In this study, the hydrolase and transferase activity of this enzyme was kinetically characterized and its potential to synthesize new fructosylated products explored. A total of 40 hydroxylated compounds were used as potential fructosyl-acceptor alternatives to sucrose. Only 17 of them, including some monosaccharides, disaccharides, and oligosaccharides as well as alditols and glycosides were fructosylated. The best alternative acceptors were the alditols. The major transfer product of the reaction including mannitol was purified and characterized as 1-O-β-D-fructofuranosyl-D-mannitol, whose maximum concentration reached 44 g/L, representing about 7.3 % of total compounds in the mixture and 89 % of all products generated by transfructosylation. The reactions including erythritol produced 35 g/L of an isomer mixture comprising 1- and 4-O-β-D-fructofuranosyl-D-erythritol. In addition, Ffase produced 24 g/L of the disaccharide blastose by direct fructosylation of glucose, which makes it the first enzyme characterized from yeast showing this ability. Thus, novel fructosylated compounds with potential applications in food and pharmaceutical industries can be obtained due to the Ffase fructosyl-acceptor promiscuity. PMID:27229725

  9. Sphingobium sp. SYK-6 LigG involved in lignin degradation is structurally and biochemically related to the glutathione transferase ω class.

    PubMed

    Meux, Edgar; Prosper, Pascalita; Masai, Eiji; Mulliert, Guillermo; Dumarçay, Stéphane; Morel, Mélanie; Didierjean, Claude; Gelhaye, Eric; Favier, Frédérique

    2012-11-16

    SpLigG is one of the three glutathione transferases (GSTs) involved in the process of lignin breakdown in the soil bacterium Sphingobium sp. SYK-6. Sequence comparisons showed that SpLigG and several proteobacteria homologues form an independent cluster within cysteine-containing GSTs. The relationship between SpLigG and other GSTs was investigated. The X-ray structure and biochemical properties of SpLigG indicate that this enzyme belongs to the omega class of glutathione transferases. However, the hydrophilic substrate binding site of SpLigG, together with its known ability to stereoselectively deglutathionylate the physiological substrate α-glutathionyl-β-hydroxypropiovanillone, argues for broadening the definition of the omega class.

  10. Involvement of glutathione transferases, Gtt1and Gtt2, with oxidative stress response generated by H2O2 during growth of Saccharomyces cerevisiae.

    PubMed

    Mariani, Diana; Mathias, Cristiane J; da Silva, Carmelita G; Herdeiro, Ricardo da Silva; Pereira, Ricardo; Panek, Anita D; Eleutherio, Elis C A; Pereira, Marcos Dias

    2008-01-01

    Glutathione transferases are detoxifying enzymes responsible for eliminating toxic compounds generated under a variety of stress conditions. Saccharomyces cerevisiae control cells and glutathione transferase mutant strains (gtt1 and gtt2) were used to analyze tolerance, lipid and protein oxidation as oxidative stress markers during growth in the presence of H2O2. Glucose 6-phosphate dehydrogenase (G6PD) and glutathione reductase were assayed to monitor the capacity of cells to recycle glutathione. Although a reduction in growth was observed, deletion of GTT1 showed less inhibition by H2O2 than the control strain. Cells showed a significant reduction in cellular viability during the first hours of growth, the gtt1 mutant being hypersensitive even after 24 h of H2O2 exposure. As a consequence of oxidative stress caused by exposure to H2O2, an increase in lipid peroxidation was observed, mainly in the glutathione transferase mutant strains. While protein carbonylation increased by 17% and 23%, respectively, after 2 h in the presence of H2O2 in the control and gtt2 mutant, a 40% increase was observed in the gtt1 strain after 24-h exposure. The antioxidant G6PD and glutathione reductase activities were affected in the gtt1 mutant during H2O2 exposure, which could be critical for recycling glutathione. The same was observed for the gtt2 mutant after 2-h treatment, indicating that glutathione recycling might be associated with the detoxification process. Thus, glutathione transferases, Gtt1 and Gtt2, seem to be crucial in the response to H2O2 stress.

  11. 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

  12. Susceptibility to endometrial cancer: influence of allelism at p53, glutathione S-transferase (GSTM1 and GSTT1) and cytochrome P-450 (CYP1A1) loci.

    PubMed Central

    Esteller, M.; García, A.; Martínez-Palones, J. M.; Xercavins, J.; Reventós, J.

    1997-01-01

    A case-control study was designed to identify associations between polymorphisms at p53, cytochrome P-450 (CYP1A1) and glutathione-S-transferases and endometrial cancer susceptibility. Among all polymorphisms analysed, an insertional variant in p53 (P53PIN3) and two polymorphisms in the 3'-end and exon 7 of CYP1A1 showed significant association with enhanced endometrial cancer risk. Images Figure 1 Figure 2 PMID:9155064

  13. 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

  14. 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. PMID:27275307

  15. 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.

  16. 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

  17. Magnesium deficiency upregulates serine palmitoyl transferase (SPT 1 and SPT 2) in cardiovascular tissues: relationship to serum ionized Mg and cytochrome c.

    PubMed

    Altura, Burton M; Shah, Nilank C; Li, Zhiqiang; Jiang, Xian-Cheng; Perez-Albela, Jose Luis; Altura, Bella T

    2010-09-01

    The present work tested the hypothesis that a short-term dietary deficiency of magnesium (Mg) (21 days) in rats would result in the upregulation of the two major subunits of serine palmitoyl-CoA-transferase, serine palmitoyl transferase (SPT 1) and SPT 2 (the rate-limiting enzymes responsible for the de novo biosynthesis of ceramides) in left ventricular, right ventricular, and atrial heart muscle and abdominal aortic smooth muscle, as well as induce a reduction in serum sphingomyelin concomitant with the release of mitochondrial cytochrome c (Cyto c) in these tissues. Our data indicate that short-term Mg deficiency (MgD) resulted in an upregulation of SPT 1 and SPT 2, concomitant with a very significant release of Cyto c in left ventricular, right ventricular, atrial, and abdominal aortic smooth muscle. Short-term MgD also produced a lowering of serum sphingomyelin and ionized Mg. The greater the reduction in serum ionized Mg, the greater the upregulation of SPT 1 and 2 and the more the increase in free Cyto c. The data suggest that MgD, most likely, causes a biosynthesis of ceramides via two pathways in cardiovascular tissues, viz., via the activation of serine palmitoyl-CoA-transferase and sphingomyelinase, which lead to apoptotic events via intrinsic (present study) and extrinsic pathways (previous studies). Low levels of drinking water Mg were cardio- and vasculoprotective.

  18. 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

  19. 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

  20. 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