Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The metaxin genes of zebrafish have been investigated by determining the sequences of metaxin cDNAs and analyzing the translated amino acid sequences. A zebrafish cDNA corresponding to a third metaxin gene was identified. Zebrafish cDNAs representing metaxins 1 and 2, previously described in human and mouse, were also identified. The zebrafish metaxin genes are designated mtx1, mtx2 and mtx3, following zebrafish nomenclature guidelines. The zebrafish metaxin 3 (ZMTX3) cDNA codes for a protein of 313 amino acids (MW 35,208), while the ZMTX1 and ZMTX2 cDNAs specify proteins of 317 residues (MW 35,906) and 274 residues (MW 30,852), respectively. Alignment of the ZMTX3 and ZMTX1 amino acid sequences revealed 40% identities, while 26% identities were found for the ZMTX3/ZMTX2 alignment. A phylogenetic tree showed that the metaxins share a common ancestry, with the grouping of the zebrafish sequences with the homologous human and mouse sequences. Analysis of the domain structure of the zebrafish metaxins uncovered a glutathione S-transferase (GST) domain for each protein and, in addition, a thioredoxin-like domain for ZMTX2. A region of transmembrane helices was found near the C-terminus for the ZMTX1 protein. In addition, regions of alpha helix were seen to be the predominant feature of zebrafish metaxin secondary structure, particularly for ZMTX2 and ZMTX3. The ZMTX3 cDNA sequence has the greatest homology to a human sequence at cytogenetic location 5q14.1, close to the thrombospondin 4 gene (THBS4). Also, the mouse metaxin 3 homologue is adjacent to Thbs4 at 13C3.
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PMID:The zebrafish metaxin 3 gene (mtx3): cDNA and protein structure, and comparison to zebrafish metaxins 1 and 2. 1508 25

Thioredoxin-binding protein-2 (TBP-2)/vitamin D(3) up-regulated protein 1 is an endogenous molecule interacting with thioredoxin (TRX), negatively regulating TRX function, and being implicated in the suppression of tumor development and metastasis. We found that TBP-2 ectopically expressed in the breast cancer cell line MCF-7 was localized predominantly in the nucleus exhibiting growth suppressive activity. The nuclear accumulation of endogenous TBP-2 protein was also demonstrated when the cells were treated with an anti-cancer drug, suberoylanilide hydroxamic acid. To investigate the mechanism underlying the nuclear localization, we performed a yeast two-hybrid screening and identified importin alpha(1) (Rch1) as a protein interacting with TBP-2. The physical interaction between TBP-2 and Rch1 was confirmed with a glutathione S-transferase pull-down assay. The interaction of TBP-2 was specific to Rch1 among other importin alpha subfamilies (Qip1 and NPI-1), and amino acids 1-227 of TBP-2 were sufficient for both the interaction with Rch1 and the nuclear localization, although there is no typical nuclear localization signal in this sequence. The expression of short interfering RNA of Rch1 suppressed suberoylanilide hydroxamic acid-induced nuclear accumulation of TBP-2. Collectively, our results strongly suggest that an interaction with importin system is required for TBP-2 nuclear translocation and growth control tightly associated with TRX-dependent redox regulation of transcription factors.
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PMID:Importin alpha1 (Rch1) mediates nuclear translocation of thioredoxin-binding protein-2/vitamin D(3)-up-regulated protein 1. 1523 75

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.
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PMID:Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli. 1535 20

The malaria parasite Plasmodium falciparum is highly adapted to cope with the oxidative stress to which it is exposed during the erythrocytic stages of its life cycle. This includes the defence against oxidative insults arising from the parasite's metabolism of haemoglobin which results in the formation of reactive oxygen species and the release of toxic ferriprotoporphyrin IX. Central to the parasite's defences are superoxide dismutases and thioredoxin-dependent peroxidases; however, they lack catalase and glutathione peroxidases. The vital importance of the thioredoxin redox cycle (comprising NADPH, thioredoxin reductase and thioredoxin) is emphasized by the confirmation that thioredoxin reductase is essential for the survival of intraerythrocytic P. falciparum. The parasites also contain a fully functional glutathione redox system and the low-molecular-weight thiol glutathione is not only an important intracellular thiol redox buffer but also a cofactor for several redox active enzymes such as glutathione S-transferase and glutaredoxin. Recent findings have shown that in addition to these cytosolic redox systems the parasite also has an important mitochondrial antioxidant defence system and it is suggested that lipoic acid plays a pivotal part in defending the organelle from oxidative damage.
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PMID:Redox and antioxidant systems of the malaria parasite Plasmodium falciparum. 1538 10

The gene coding for glutathione S-transferase (GST) has been isolated from the Mytilus edulis hepatopancreas. Open reading frame analysis indicated that the M. edulis GST (meGST) gene encodes a protein of 206 amino acid residues with a calculated molecular mass of 23.68 kDa. The deduced amino acid sequence showed high sequence similarity with the sequence of the pi class GST. The meGST was expressed in Escherichia coli, and the recombinant meGST was purified by affinity chromatography and characterized. The recombinant meGST exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). Kinetic analysis with respect to CDNB as substrate gave a K(m) of 0.68 mM and a V(max) of 0.10 mmol/min per mg protein. The recombinant meGST had a maximum activity at approximately pH 8.5, and its optimum temperature was 39 degrees C. The predicted three-dimensional structure of the meGST revealed the N-terminal domain possesses a thioredoxin fold and the six helices of the C-terminal domain make a alpha-helical bundle. These features indicate that the meGST belongs to pi class GST.
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PMID:Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis. 1546 63

Glutathione peroxidase (GPx, EC 1.11.1.9) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several attempts have been made to imitate its function for mechanical study and for its pharmacological development as an antioxidant. By replacing the active site serine 9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione S-transferase (GST, EC 2.5.1.18) from Lucilia cuprina was converted into a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPx in the specific GSH binding sites and the geometric conformation for the active selenocysteine in their common GSH binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPxs. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPx. It exhibited pH and temperature-dependent catalytic activity and a typical ping-pong kinetic mechanism. Engineering GST into an efficient GPx-like biocatalyst provided new proof for the previous assumption that both GPx and GST were evolved from a common thioredoxin-like ancestor to accommodate different functions throughout evolution.
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PMID:Engineering glutathione transferase to a novel glutathione peroxidase mimic with high catalytic efficiency. Incorporation of selenocysteine into a glutathione-binding scaffold using an auxotrophic expression system. 1564 95

Ixodes ricinus L. is the principal European vector of Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. Subtractive hybridization was used to isolate tick genes that were induced in whole ticks after blood meals on uninfected and B. burgdorferi-infected guinea pigs. Novel cDNA clones with similarity to cytochrome c oxidase, salivary secreted protein, actin, and a cysteine protease propeptide were induced after a blood meal. Novel cDNA clones with similarity to thioredoxin peroxidases, dolichyl-phosphate beta-glucosyltransferase, glutathione S-transferase, defensin, ML domain-containing protein, and von Willebrand factor were induced after B. burgdorferi infection. Virtual Northern analysis was used to verify that these genes were differentially expressed in ticks after a pathogen-infected blood meal and to detect their tissues of expression. The characterization of genes that are induced after an infected blood meal is essential for gaining an understanding of the molecular mechanisms that underlie vector-pathogen interactions.
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PMID:Differential expression of Ixodes ricinus tick genes induced by blood feeding or Borrelia burgdorferi infection. 1569 Oct 6

Transforming growth factor beta (TGF-beta) stimulation results in the assembly of Smad-containing protein complexes that mediate activation or repression of TGF-beta responsive genes. To determine if disruption of specific Smad protein-protein interactions would selectively inhibit responses to TGF-beta or generally interfere with Smad-dependent signaling, we developed three Smad-binding peptide aptamers by introducing Smad interaction motifs from Smad-binding proteins CBP, FoxH1 and Lef1 into the scaffold protein E. coli thioredoxin A (Trx). All three classes of aptamers bound to Smads by GST pulldown assays and co-immunoprecipitation from mammalian cells. Expression of the aptamers in HepG2 cells did not generally inhibit Smad-dependent signaling as evaluated using seven TGF-beta responsive luciferase reporter genes. The Trx-xFoxH1b aptamer inhibited TGF-beta-induced expression from a reporter dependent on the Smad-FoxH1 interaction, A3-lux, by 50%. Trx-xFoxH1b also partially inhibited two reporters not dependent on a Smad-FoxH1 interaction, 3TP-lux and Twntop, and endogenous PAI-1 expression. Trx-Lef1 aptamer only inhibited expression of the Smad-Lef1 responsive reporter gene TwnTop. The Trx-CBP aptamer had no significant effect on reporter gene expression. The results suggest that Smad-binding peptide aptamers can be developed to selectively inhibit TGF-beta-induced gene expression.
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PMID:Selective inhibition of TGF-beta responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP. 1575 Jun 22

Thiol proteins are important in cellular antioxidant defenses and redox signalling. It is postulated that reactive oxidants cause selective thiol oxidation, but relative sensitivities of different cell proteins and critical targets are not well characterized. We exposed Jurkat cells to H2O2 for 10 min and measured changes in reversibly oxidized proteins by labelling with iodoacetamidofluorescein and two-dimensional electrophoresis. At 200 microM H2O2, which caused activation of the MAP (mitogen-activated protein) kinase ERK (extracellular-signal-regulated kinase), growth arrest and apoptosis, relatively few changes were seen. A total of 28 spots were reversibly oxidized (increased labelling intensity) and 24 decreased. The latter included isoforms of peroxiredoxins 1 and 2, which were irreversibly oxidized. Oxidation of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was striking, and other affected proteins included glutathione S-transferase P1-1, enolase, a regulatory subunit of protein kinase A, annexin VI, the mitotic checkpoint serine/threonine-protein kinase BUB1beta, HSP90beta (heat-shock protein 90beta) and proteosome components. At 20 microM H2O2, changes were fewer, but GAPDH and peroxiredoxin 2 were still modified. Dinitrochlorobenzene treatment, which inhibited cellular thioredoxin reductase and partially depleted GSH, caused reversible oxidation of several proteins, including thioredoxin 1 and peroxiredoxins 1 and 2. Most changes were distinct from those with H2O2, and changes with H2O2 were scarcely enhanced by dinitrochlorobenzene. Relatively few proteins, including deoxycytidine kinase, nucleoside diphosphate kinase and a proteosome activator subunit, responded only to the combined treatment. Thus most of the effects of H2O2 were not linked to thioredoxin oxidation. Our study has identified peroxiredoxin 2 and GAPDH as two of the most oxidant-sensitive cell proteins and has highlighted how readily peroxiredoxins undergo irreversible oxidation.
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PMID:Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells. 1580 6

Glutaredoxin-like proteins form a new subgroup of glutaredoxins with a serine replacing the second cysteine in the CxxC-motif of the active site. Yeast Grx5 is the only glutaredoxin-like protein studied biochemically so far. We identified and cloned three genes encoding glutaredoxin-like proteins from the malaria parasite Plasmodium falciparum (Pf Glp1, Pf Glp2, and Pf Glp3) containing a conserved cysteine in the CGFS-, CKFS-, and CKYS-motif, respectively. Here, we describe biochemical properties of Pf Glp1 and Pf Glp2. Cys 99, the only cysteine residue in Pf Glp1, has a pK(a) value as low as 5.5 and is able to mediate covalent homodimerization. Monomeric and dimeric Pf Glp1 react with GSSG and GSH, respectively. Pf Glp2 is monomeric and both of its cysteine residues can be glutathionylated. Molecular models reveal a thioredoxin fold for the putative C-terminal domain of Pf Glp1, Pf Glp2, and Pf Glp3, as well as conserved residues presumably required for glutathione binding. However, Pf Glp1 and Pf Glp2 neither possess activity in a classical glutaredoxin assay nor display activity as glutathione peroxidase or glutathione S-transferase. Mutation of Ser 102 in the CGFS-motif of Pf Glp1 to cysteine did not generate glutaredoxin activity either. We conclude that, despite their ability to react with glutathione, glutaredoxin-like proteins are a mechanistically and functionally heterogeneous group with only little similarities to canonical glutaredoxins.
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PMID:Plasmodium falciparum glutaredoxin-like proteins. 1584 45


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