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Gestational diabetes (GD) is a condition defined as carbohydrate intolerance and hyperglycemia beginning in the second trimester of pregnancy, which overlaps with the progesterone exponential increase. Progesterone has been shown to cause pancreatic β-cell death by a mechanism dependent on the generation of reactive oxygen species and oxidative stress. Herein, we studied the effect of this hormone on the expression of 84 genes related to oxidative stress and oxidant defense in pancreatic RINm5F cell lineage. Cells were incubated with 0.1, 1.0, or 100 μM progesterone for 6 or 24 h, in the presence or absence of the vitamins E and C. Among the investigated genes, five of them had their expression increased, at least 2-fold, in two different concentrations independently of the time of incubation, or at the same concentration at the different time points, including those that encode for stearoyl-CoA desaturase 1 (Scd1), dual oxidase 1 (Duox1), glutathione peroxidase 6 (GPx6), heme oxygenase 1 (Hmox1), and heat shock protein a1a (Hspa1a). Vitamins E and C were able to increase, in progesterone-treated cells, the expression of genes with antioxidant function such as Hmox1, but decreased Scd1 expression, a gene with prooxidant function. At cytoplasmic level, progesterone positively modulated Hmox1 and Hspa1a content. These results suggest that the protein encoded by these genes might protect cells against progesterone induced-oxidative damage, opening perspectives to elucidate the molecular mechanism involved in progesterone action in GD, as well as for the development of antioxidant strategies for the prevention and treatment of this disease.

The aim of the present study was to analyze the selenoprotein expression levels in gastric cancer patients. We enrolled 40 patients (29 males, 11 females) who were recently diagnosed with gastric cancer and 50 healthy people (30 males, 20 females) as controls. The expression of 25 selenoprotein genes (Dio1, Dio2, Dio3, Gpx1, Gpx2, Gpx3, Gpx4, Gpx6, SelH, SelI, SelK, SelM, SelN, SelO, SelP, SelS, SelT, SelV, SelW, SelX, Sel15, Sps2, TR1, TR2, and TR3) in human gastric cancer tissues, para-carcinoma tissues, adjacent normal gastric tissues, erythrocytes, and lymphocytes in the gastric cancer group and healthy control group was analyzed by qRT-PCR. Here, we showed that among the 25 selenoproteins, 13 selenoproteins in erythrocytes (Gpx1, Gpx4, Sel15, TR1, TR2, SelH, SelK, SelM, SelO, SelS, SelV, SelW, and Sps2), 15 selenoproteins in lymphocytes (Gpx1, Gpx4, Sel15, TR1, TR2, SelH, SelK, SelN, SelO, SelS, SelT, SelV, SelX, SelW, and Sps2) and 13 selenoproteins in gastric cancer and para-carcinoma tissues (Dio1, Dio2, Dio3, Gpx1, Gpx4, Sel15, SelH, SelK, SelM, SelS, SelT, SelW, and Sps2) were significantly decreased (P

The effect of 3,5-dicarbomethoxyphenylbiguanide, which was selected with the Prediction of Activity Spectra of Substances (PASS) computer program, on the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione transferase in the heart and the blood serum of rats with experimental rheumatoid arthritis was investigated. The studied parameters changed towards control values when the tested compound was injected in animals with the pathology. These results can be explained by the cardioprotective and antioxidant activity of the compound. The data obtained during the study may be used for the development of new preventive and therapeutic agents for the treatment of the rheumatoid arthritis.

Background Selenium (Se) is an essential micronutrient required by avian species. Dietary Se/vitamin E deficiency induces three classical diseases in chicks: exudative diathesis, nutritional pancreatic atrophy, and nutritional muscular dystrophy. Scope of review This review is to summarize and analyze the evolution, regulation, and function of avian selenogenome and selenoproteome and their relationship with the three classical Se/vitamin E deficiency diseases. Major conclusions There are 24 selenoproteins confirmed in chicks, with two avian-specific members (SELENOU and SELENOP2) and two missing mammalian members (GPX6 and SELENOV). There are two forms of SELENOP containing 1 or 13 selenocysteine residues. In addition, a Gallus gallus gene was conjectured to be the counterpart of the human SEPHS2. Expression of selenoprotein genes in the liver, pancreas, and muscle of chicks seemed to be highly responsive to dietary Se changes. Pathogeneses of the Se/vitamin E deficient diseases in the chicks were likely produced by missing functions of selected selenoproteins in regulating cellular and tissue redox balance and inhibiting oxidative/reductive stress-induced cell death. General significance Gene knockout models, similar to those of rodents, will help characterize the precise functions of avian selenoproteins and their comparisons with those of mammalian species.

Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Congenital toxoplasmosis may result in abortion, severe mental retardation and neurologic damage in the offspring. Placental damage is considered as the key event in this disease. Here we show that maternal infection with Toxoplasma gondii Wh3 isolate of genotype Chinese 1, which is predominantly prevalent in China, induced trophoblast apoptosis of pregnant mouse. PCR array analysis of 84 key genes in the biogenesis and functions of mouse mitochondrion revealed that ten genes were up-regulated at least 2-fold in the Wh3 infection group, compared with those in the control. The elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG), as well as the decreased glutathione (GSH), were observed in the infected mice. The mRNA levels of NADPH oxidase 1 and glutathione peroxidase 6 (GPx6) were significantly increased. The production of excessive ROS was NADPH oxidase-dependent, which contributed to mitochondrial structural damage and mitochondrial dysfunction in placentas, followed by the cleavage of caspase-9 and caspase-3, and finally resulted in apoptosis of trophoblasts. All the above-mentioned phenomena were inhibited by pretreatment with the antioxidant of N-acetylcysteine (NAC). Taken together, we concluded that Wh3 infection during pregnancy may contribute to trophoblast apoptosis by oxidative stress-induced mitochondrial dysfunction and activation of the downstream signaling pathway.

In this study, proteome and activities of glutathione (GSH)-related enzymes were investigated in detached leaves of Arabidopsis thaliana treated with ochratoxin A (OTA) alone or supplemented with buthionine sulfoximine (BSO, a specific inhibitor of the first step in GSH biosynthesis). A comparative proteomic study using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS) identified 12 differentially expressed proteins mainly involved in GSH metabolism, energy metabolism, sugar metabolism, and photosynthesis. The treatment with OTA significantly enhanced the activities of glutathione-S-transferase (GST) and glutathione reductase (GR) through up-regulating the corresponding genes (GSTF7, GR1), an the diminishing effect of BSO on them counteracted the results. However, both OTA and BSO decreased the activity of ascorbate peroxidase (APX), and OTA also decreased the monodehydroascorbate reductase (MDHAR) and glutathione peroxidase (GPX) activities. Briefly, the OTA-induced phytotoxicity to the A. thaliana detached leaves was increased slightly by addition of BSO, and the fluctuation in GSH synthesis, GSH metabolism and disorder of cellular metabolism happened.

SummaryHow mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a reactive oxygen species scavenging enzyme glutathione peroxidase 1. Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth.

The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNAUTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNAUTu and seven chimeric tRNAs that were constructed on the basis of tRNAUTu. We found that chimeric tRNAUTu2, which substitutes the acceptor stem and T-stem of tRNAUTu with those from tRNASec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNAUTuT6, which has a single base change (A59C) compared to tRNAUTu, mediated the highest reactive expression of hGPx1. ...

Glutathione peroxidase is key for the removal of H2O2 and other hydroperoxides and therefore, it has an important role in the maintenance of the reactive oxygen species (ROS) metabolic balance in vivo. The native enzymes of the glutathione peroxidase family (GPx) have many defects, such as instability in vitro and poor availability. GPx mimetics has become a topic of considerable interest in artificial enzyme research. Many forms of GPx mimics have been synthesized, by including selenium and tellurium (double-bridged and single-bridged, 2-substituted and 6-substituted) in a mother molecule but differences the GPx mimics enzymatic activity have rarely been compared. We designed and synthesized eight cyclodextrin derivatives and used two types of enzyme assays to determine their activities. The results show that: (a) tellurium-containing GPx mimics have higher activity than that of selenium-containing GPx mimics; (b) dual-bridged mimics have higher activity than bis-bridged mimics; and (c) 2-position modified cyclodextrin has higher activity than 6-position modified cyclodextrin.

A full-length cDNA clone encoding an 866 bp-length glutathione peroxidase protein (NnGPX) was isolated from lotus (Nelumbo nucifera L.). The deduced amino acid sequence of the NnGPX gene had significant homology with ATGPX6. A 3D structural model of the NnGPX was constructed by homology modeling. The cloned NnGPX gene was expressed in Escherichia coli, and a fusion protein of about 40 kDa was detected after isopropyl thiogalactoside induction. Under different concentrations of Na2SeO3 treatments, NnGPX was found to be an enzyme that does not contain selenium. Real-time PCR analysis showed that the NnGPX gene was expressed in all organs of lotus, and its high expression mainly occurred in organs with active metabolisms. NnGPX transcript increased remarkably in response to cold, heat, mechanical damage, and salt treatment. Subsequently, the NnGPX gene was introduced in Oryza sativa cv. Yuetai B. PCR results verified the integration of this gene into the genome of rice and reverse transcription-PCR verified that this gene had been expressed in transgenic rice. The transgenic plants were significantly more tolerant to salt stress compared with the wild-type.

The free radicals within the body, produced by metabolic activities or derived from environmental sources are relatively related to hepatoxicity. Since heavy metals including iron have the ability to produce free radicals, the liver glutathione system neutralizes them to protect cells against any damage. The objective of this study is to indicate the toxic effects of iron on the glutathione system at the enzymatic and molecular level. Thus, any possible correlation between enzymatic and molecular levels can be determined. According to our results, while mRNA expression of glutathione reductase (Gsr) and glutathione S-transferases (Gsta5) genes were not affected by long-term exposure to various concentrations of iron (Fe3+), transcription level of glutathione peroxidase (Gpx2) was influenced in the presence of toxic iron. Whereas the enzyme activites of GSR (GR), GPX and GST were significantly affected in rat liver.

Background/Aims: Altered expression and activity of the antioxidant enzymes glutathione peroxidases (GPXs) have been observed in obesity in human and animal studies. We aimed to study 59 single nucleotide polymorphisms (SNPs) for GPX1-7 genes and to characterize their association with prepubertal childhood obesity and its associated biomarkers. Methods: This case-control study included 193 obese and 191 normal-weight prepubertal Spanish children, in whom anthropometry, biochemical parameters, adipokines, antioxidant enzyme erythrocyte activities and biomarkers of oxidative stress, inflammation and cardiovascular risk were measured. The genotype analysis was performed in the Illumina platform. PLINK and SPSS were used for statistical analyses. Results: We found SNPs rs757228 and rs8103188 (GPX4) to be negatively associated and rs445870 (GPX5) and rs406113 (GPX6) to be positively associated with obesity in children. The variant rs2074451 (GPX4) increased GPX activity in erythrocytes. Although we did not find significant differences in erythrocyte GPX activity between obese and normal-weight children, GPX activity was found to be positively and significantly correlated with blood pressure, adipocyte fatty acid-binding protein and high-sensitivity C-reactive protein. Conclusions: The GPX variants rs757228, rs8103188, rs445870 and rs406113 were associated with prepubertal childhood obesity. However, erythrocyte GPX activity was not altered in obese prepubertal Spanish children.

Glutathione peroxidase (GPX) is one of the key enzymes that protect cells against oxidative damage caused by reactive oxygen species. Previous studies of plant GPXs focused mainly on angiosperms. In contrast, little information is available on the molecular characteristics of this gene family in gymnosperms. In this study, four GPX genes (PtaGPX1, 2, 3, and 4) were cloned from the gymnosperm Pinus tabulaeformis, which showed high protein sequence identity and similar expression patterns in various tissues. The four Pinus GPX proteins were expressed in Escherichia coli, and the purified proteins used thioredoxin, but not glutathione, as an electron donor. The four Pinus GPXs showed different enzymatic activities and kinetic characteristics, suggesting functional divergence. Two conserved Cys residues (corresponding to Cys44 and Cys92 of PtaGPX3) were identified in all plant GPXs, and their functions were assessed using site-directed mutagenesis. Cys44 and Cys92 of PtaGPX3 could form an intramolecular disulfide bond under oxidizing conditions. These two residues were critical components of active sites and contributed to catalytic activity. This study provides novel insights into the functional divergence and catalytic properties of the GPX family in gymnosperms.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC1.11.1.12) is a selenoperoxidase that can directly reduce phospholipid and cholesterol hydroperoxides. The mature cytoplasmic GPx4 is a monomeric protein with molecular weight of 19.5 kDa. In this study, human GPx4 (hGPx4) gene was amplified from the complementary DNA library of human hepatoma cell line. Eukaryotic expression plasmid pSelExpress1-leader-GPx4 was constructed and transfected into the eukaryotic cells HEK293T. Expression of hGPx4 was detected by Western blotting, and the target protein was purified by immobilized metal affinity chromatography. The results of the activity and kinetics of the purified protein show that the obtained protein follows a "ping-pong" mechanism, which is similar to that of native cytosolic glutathione peroxidase (GPx1; EC1.11.1.9). This is the first time that hGPx4 could be expressed and purified from HEK293T cells, and this work will provide an important resource of hGPx4 for its functional study in vitro and in vivo.

A selenium-dependent glutathione peroxidase cDNA was obtained from the ridgetail white prawn Exopalaemon carinicauda ( EcGPx ) by rapid amplification of cDNA ends (RACE) methods. The full-length cDNA of EcGPx was 946 bp, which contains a 5′-untranslated region (UTR) of 105 bp, 3′-UTR of 280 bp with a poly (A) tail, an open reading frame (ORF) of 561 bp, encoding a 186 amino-acid polypeptide with the predicted molecular weight of 21.35 kDa and estimated isoelectric point of 7.65. It involves a putative selenocysteine (U 39 ) residue which is encoded by an opal codon, 220 TGA 222 , and forms an active site with residues Q 73 and W 141 . Sequence analysis revealed that a GPx signature motif 2 ( 63 LAFPCNQF 70 ), an extra active site motif ( 151 WNFEKF 156 ), two putative N-glycosylation site ( 75 NNT 77 and 107 NGS 109 ), and two arginine residues (R 89 and R 167 ) were observed in the EcGPx sequence. Comparison of amino acid sequences showed that white shrimp GPx is more closely related to GPx1 and GPx2 subgroups. Quantitative real-time RT-PCR analysis indicated that two glutathione antioxidant enzymes of E. carinicauda , glutathione peroxidase (designated EcGPx ) and glutathione S-transferase (designated EcGST ) were widely expressed in all the tested tissues, but showed different expression patterns. After Vibrio anguillarum and WSSV challenge, EcGPx and EcGST transcripts both in hemocytes and hepatopancreas increased in the first 6 h and 3 h, respectively. The results suggested that EcGPx and EcGST might be associated with the immune defenses to V. anguillarum and WSSV in E. carinicauda .

Glutathione is the most abundant nonprotein molecule in the cell and plays an important role in many biological processes, including the maintenance of intracellular redox states, detoxification, and metabolism. Furthermore, glutathione levels have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer. A novel concept in bioanalysis is introduced and applied to the highly sensitive and inexpensive detection of reduced glutathione (GSH), over its oxidized form (GSSG), and glutathione reductase (GR) in human serum. This new fluorogenic bioanalytical system is based on the GSH-mediated stabilization of growing CdS nanoparticles. The sensitivity of this new assay is 5 pM of GR, which is 3 orders of magnitude better than other fluorogenic methods previously reported.

Glutathione peroxidase (GPx) and glutathione reductase (GR) are key enzymes in the antioxidant defense systems of living organisms and protect organisms from environmental stresses. To investigate the mechanism of resistance to heat stress in the red alga Gracilaria lemaneiformis, the cDNA sequences encoding glutathione peroxidase and glutathione reductase of two strains (wild type and heat-tolerant cultivar 981) of G. lemaneiformis were successfully cloned using reverse transcription PCR and rapid amplification cDNA ends techniques. cDNA encoding GPx has 930 nucleotides with an open reading frame (ORF) of 750 nucleotides, encoding 249 amino acid residues. cDNA encoding GR was 1,572 nucleotides with a 1,416-nucleotide ORF encoding 471 amino acids. No introns existed in genomic DNA of GPx and GR of G. lemaneiformis. Southern blotting indicated that there was only one copy of GPx and GR in both strains. Under heat stress, bioactivity and mRNA transcription levels of both GPx and GR were upregulated in cultivar 981 compared to those in the wild type. These results indicate that both GPx and GR may play important roles in the ability of G. lemaneiformis to resist high temperatures.

Px IV is a distant relative of the known glutathione peroxidase-type enzymes of African trypanosomes. Immunofluorescence microscopy of bloodstream cells expressing C-terminally Myc6-tagged Px IV revealed a mitochondrial localization. Recombinant Px IV possesses very low activity as glutathione peroxidase but catalyzes the trypanothione/tryparedoxin-dependent reduction of hydrogen peroxide and, even more efficiently, of arachidonic acid hydroperoxide. Neither overexpression in bloodstream cells nor the deletion of both alleles in bloodstream or procyclic parasites affected the in vitro proliferation. Trypanosoma brucei Px IV shares 58% of all residues with TcGPXII. The orthologous enzymes have in common their substrate preference for fatty acid hydroperoxides. However, the T. cruzi protein has been reported to be localized in the endoplasmic reticulum and to be specific for glutathione as reducing agent. Taken together, our data show that Px IV is a low abundant tryparedoxin peroxidase of T. brucei that is not essential, at least under culture conditions.

Glutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ∼50% sequence identity. Human and mouse ChaC2 proteins purified in vitro show 10–20-fold lower catalytic efficiency than ChaC1, although they showed comparable Km values (Km of 3.7 ± 0.4 mm and kcat of 15.9 ± 1.0 min−1 toward glutathione for human ChaC2; Km of 2.2 ± 0.4 mm and kcat of 225.2 ± 15 min−1 toward glutathione for human ChaC1). The ChaC1 and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either γ-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed.

The glutathione-related enzymes are usually considered to accompany the main non-enzymatic antioxidative compounds of the ascorbate–glutathione cycle. Taking into account that the redox processes are not spontaneous in cells, but the adequate reaction velocity and appropriate specificity are achieved by the catalyzing activity of enzymes, special attention has raised toward the glutathione-utilizing enzymes. Glutathione reductase (GR) is a NADPH-dependent oxidoreductase which catalyzes the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH). Some members of the diverse glutathione transferase (GST) enzyme family have GSH-dependent thiol transferase activity and participate in the recycling of antioxidants (ascorbate, flavonoids, quinones), while other isoenzymes, due to their S-transferase activity, are involved in the detoxification mechanisms using GSH as co-substrate. A significant portion of GST isoenzymes also has glutathione peroxidase activity and can convert lipid peroxides and other peroxides to less harmful compounds. The plant glutathione peroxidase enzymes (GPXs) may be involved in the detoxification of H2O2 and organic hydroperoxides and in the regulation of the cellular redox homeostasis by maintaining the thiol/disulfide balance. Most of plant GPXs prefer to use thioredoxin (TRX) instead of glutathione as a reducing agent, and it is thought that the GPXs may represent a link between the glutathione- and the thioredoxin-based system. The GR, GPX and some GST isoenzymes have Cys in their active center and thus are directly regulated by redox status. This chapter summarizes their roles in stress responses as antioxidant enzymes, in determining the redox status of cells, and emphasizes their connection to redox signaling mechanisms.

We studied phospholipid composition of erythrocytes and the state of the glutathione redox system in rats during adaptation to cholesterol load for 180 days. The adaptive response is formed during the period from day 30 to day 90 of cholesterol load and is associated with increased relative content of phosphatidylethanolamine and phosphatidylinositol, activation of glutathione redox system enzymes (glutathione reductase and glutathione peroxidase), and enhanced production of glutathione.

Glutathione peroxidases (Gpxs) are the key anti-oxidant enzymes found in Saccharomyces cerevisiae. Among the three Gpx isoforms, glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and modulates the activities of redox-sensitive thiol proteins involved in various biological reactions. By using a proteomic approach, glyceralde-hyde-3-phosphate dehydrogenase 2 (GAPDH2; EC 1.2.1.12) was found as a candidate protein for interaction with Gpx3. GAPDH, a key enzyme in glycolysis, is a multi-functional protein with multiple intracellular localizations and diverse activities. To validate the interaction between Gpx3 and GAPDH2, immunoprecipitation and a pull-down assay were carried out. The results clearly showed that GAPDH2 interacts with Gpx3 through its carboxyl-terminal domain both in vitro and in vivo. Additionally, Gpx3 helps to reduce the S-nitrosylation of GAPDH upon nitric oxide (NO) stress; this subsequently increases cellular viability. On the basis of our findings, we suggest that Gpx3 protects GAPDH from NO stress and thereby contributes to the maintenance of homeostasis during exposure to NO stress.

There is much information about glutathione (GSH) in eukaryotic cells, but relatively little is known about GSH in prokaryotes. Without GSH and glutathione redox cycle lactic acid bacteria (LAB) cannot protect themselves against reactive oxygen species. Previously we have shown the presence of GSH in Lactobacillus fermentum ME-3 (DSM14241). Results of this study show that probiotic L. fermentum ME-3 contains both glutathione peroxidase and glutathione reductase. We also present that L. fermentum ME-3 can transport GSH from environment and synthesize GSH. This means that it is characterized by a complete glutathione system: synthesis, uptake and redox turnover ability that makes L. fermentum ME-3 a perfect protector against oxidative stress. To our best knowledge studies on existence of the complete glutathione system in probiotic LAB strains are still absent and glutathione synthesis in them has not been demonstrated.

The budding yeast Saccharomyces cerevisiae has three homologues of glutathione peroxidase (GPX1, GPX2, and GPX3). Two structural homologues of the mammalian glutathione peroxidase, Gpx2 and Gpx3, have been proven to be atypical 2-Cys peroxiredoxins, which prefer to use thioredoxin as an electron donor. Here, we show that Gpx1 is also an atypical 2-Cys peroxiredoxin, but uses glutathione and thioredoxin almost equally. We determined the redox state of Gpx1 in vivo.

Oxidative stress in plants causes the induction of several enzymes, including superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The first two are responsible for converting superoxide to H2O2 and its subsequent reduction to H 2O, and the third is involved in recycling of ascorbate. Glutathione peroxidases (GPXs, EC 1.11.1.9) are a family of key enzymes involved in scavenging oxyradicals in animals. Only recently, indications for the existence of this enzyme in plants were reported. Genes with significant sequence homology to one member of the animal GPX family, namely phospholipid hydroperoxide glutathione peroxidase (PHGPX), were isolated from several plants. In this paper we report the homology modelling of the glutathione peroxidase protein from Ricinus communis L. and its interactions with its two substrates hydrogen peroxide and glutathione. Specific sites of interaction were identified and ligand binding pockets were also screened.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC 1.11.1.12) is an antioxidant enzyme that reduces lipid hydroperoxides in biomembranes. Here, we cloned and characterized cys-PHGPx from the bumblebee Bombus ignitus (Bi-PHGPx). The Bi-PHGPx gene consists of 4 exons, encoding 168 amino acid residues with a canonical cys-codon at residue 45 and active site residues Gln(82) and Trp(134). Recombinant Bi-PHGPx, expressed as a 19 kDa protein in baculovirus-infected insect cells, exhibited enzymatic activity against PLPC-OOH and H(2)O(2) using glutathione as an electron donor. Tissue distribution analyses showed the presence of Bi-PHGPx in all tissues examined. Bi-PHGPx transcripts were upregulated by stresses, such as wounding, H(2)O(2) exposure, external temperature shock, and starvation. Under H(2)O(2) overload, the RNA interference (RNAi)-induced thioredoxin peroxidase (BiTPx1)-knock-down B. ignitus worker bees showed upregulated expression of Bi-PHGPx in the fat body. These results indicate that Bi-PHGPx is a stress-inducible antioxidant enzyme that acts on phospholipid hydroperoxide and H(2)O(2).

When hemolyzates from erythrocytes of selenium-deficient rats were incubated in vitro in the presence of ascorbate or H(2)O(2), added glutathione failed to protect the hemoglobin from oxidative damage. This occurred because the erythrocytes were practically devoid of glutathione-peroxidase activity. Extensively purified preparations of glutathione peroxidase contained a large part of the (75)Se of erythrocytes labeled in vivo. Many of the nutritional effects of selenium can be explained by its role in glutathione peroxidase.

A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.

In order to generate catalytic antibodies with glutathione peroxidase (GPX) activity, we prepared GSH-S-2,4-dinitrophenyl t-butyl ester (GSH-S-DNPBu) as target antigen. Three clones (A11, B3, and D5) that bound specifically to the antigen were selected from the phage display antibody library (human synthetic VH + VL single-chain Fv fragment (scFv) library). Analysis of PCR products using gel electrophoresis and sequencing showed that only clone B3 beared intact scFv-encoding gene, which was cloned into the expression vector pPELB and expressed as soluble form (scFv-B3) in Escherichia coli Rosetta. The scFv-B3 was purified by Ni(2+)-immobilized metal affinity chromatography (IMAC). The yield of purified proteins was about 2.0-3.0 mg of proteins from 1 L culture. After the active site serines of scFv-B3 were converted into selenocysteines (Secs) with the chemical modification method, we obtained the human catalytic antibody (Se-scFv-B3) with GPX activity of 1288 U/micromol.

Glutathione peroxidases (EC 1.11.1.9 and EC 1.11.1.12) catalyze the reduction of H(2)O(2) or organic hydroperoxides to water or corresponding alcohols using reduced glutathione. Some glutathione peroxidase isozymes have a selenium-dependent glutathione peroxidase activity and present a selenocysteine encoded by the opal TGA codon. In the present study, insights into the evolution of the whole glutathione peroxidase gene family were obtained after a comprehensive phylogenetic analysis using the improved number of glutathione peroxidase sequences recorded in the PeroxiBase database (http://peroxidase.isb-sib.ch/index.php). The identification of a common ancestral origin for the diverse glutathione peroxidase clusters was not possible. The complex relationships and evolutionary rates of this gene family suggest that basal glutathione peroxidase classes, present in all kingdoms, have originated from independent evolutionary events such as gene duplication, gene losses, lateral gene transfer among invertebrates and vertebrates or plants. In addition, the present study also emphasizes the possibility of some members being submitted to strong selective forces that probably dictated functional convergences of taxonomically distant groups.

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 μm native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme–electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.

The genome sequence of Schizosaccharomyces pombe reveals only one gene for a putative glutathione peroxidase (gpx1(+)). The Gpx1 protein has a peroxidase activity but preferred thioredoxin to glutathione as an electron donor when examined in vitro and in vivo, and therefore is a thioredoxin peroxidase. Besides H(2)O(2), it can reduce alkyl and phospholipid hydroperoxides. Expression of the gpx1 gene was elevated at the stationary phase, and we found that it supported long-term survival of S. pombe. The mutant also exhibited some defect in the activity of aconitase, an oxidation-labile Fe-S enzyme in mitochondria. Activity of sulfite reductase, a labile Fe-S enzyme in the cytosol, was also dramatically lowered in the mutant in the stationary phase. The Gpx1 protein, without any obvious targeting sequence, was localized in mitochondria as well as in the cytosol. Therefore, Gpx1 must serve to ensure optimal mitochondrial function and cytosolic environment, especially in the stationary phase.

The aim of the study was to establish a 96-well microtiter plate-based reporter gene assay to test the influence of natural compounds on the promoter activities of rat catalase, human glutathione peroxidase and human superoxide dismutase expressed in V79 cells. Luciferase expression vectors with the promoter regions of the genes coding for the three above-mentioned enzymes were constructed and transfected into V79 cells. Thereafter the ability of sodium ascorbate, L-carnitine, catechin, epigallocatechin gallate, genistein, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate and Trolox to enhance the promoter activities was evaluated. Genistein, paraquat and quercetin led to a statistically significant increase in the glutathione peroxidase and superoxide dismutase gene promoter activities. None of the compounds tested enhanced the catalase gene promoter activity. The reporter gene assay described in this report is easy to perform, fast and allows one to test a high number of compounds and different concentrations of a single compound at the same time.

The redox enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) has emerged as one of the most significant selenoenzymes in mammals, corroborated by early embryonic lethality of PHGPx null mice. PHGPx is one of five selenium-dependent glutathione peroxidases and the second glutathione peroxidase to be discovered in 1982. PHGPx has a particular position within this family owing to its peculiar structural and catalytic properties, its multifaceted roles during male gametogenesis, and its necessity for early mouse development. Interestingly, mice devoid of endogenous glutathione die at the same embryonic stage as PHGPx-deficient mice compatible with the hypothesis that a similar phenotype of embryonic lethality may be provoked by PHGPx deficiency and lack of its reducing substrate glutathione. Various gain- and loss-of-function approaches in mice have provided some insights into the physiological functions of PHGPx. These include a protective role for PHGPx in response to irradiation, increased resistance of transgenic PHGPx mice to toxin-induced liver damage, a putative role in various steps of embryogenesis, and a contribution to sperm chromatin condensation. The expression of three forms of PHGPx and early embryonic lethality call for more specific studies, such as tissue-specific disruption of PHGPx, to precisely understand the contribution of PHGPx to mammalian physiology and under pathological conditions.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% alpha-helix, 30.7% beta-sheet, 18.5% gamma-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27 degrees C. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and H(2)O(2). These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.

Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis.

Objectives : This study was carried out to examine protective effect of wild ginseng extract on HepG2 human hepatoma cell line against tert-Butyl hydroperoxide (t-BHP)-induced oxidative damage. Methods : To evaluate protective effect of wild ginseng extract against t-BHP induced cytotoxicity, LDH level and activity of glutathione peroxidase and reductase were measured. Gene expression was also measured using DNA microarray. Results : Wild ginseng extract showed a significant protective effect against t-BHP-induced cytotoxicity in HepG2 cell line. It is not, however, related with the activities of glutathione peroxidase and glutathione reductase. Analysis of gene expression using DNA chip, demonstrated that 28 genes were up-regulated in t-BHP only group. Five genes - selenoprotein P, glutathione peroxidase 3, sirtuin 2, peroxiredoxin 2, serfiredoxin 1 homolog - may be related with the protective effect of wild ginseng extract. Conclusions : Based on the results, a protective effect of wild ginseng extract against t-BHP-induced oxidative damage in HepG2 cell line is not associated with the activities of glutathione peroxidase and glutathione reductase, but with the expression of selenoprotein P, glutathione peroxidase 3, sirtuin 2, peroxiredoxin 2, and serfiredoxin 1 homolog.

The activities of Se-dependent glutathione peroxidases and non-Se-dependent glutathione peroxidase in the submandibular gland were observed using specific substrates. The activities for H2O2, cumene hydroperoxide, tert-butyl hydroperoxide, and phosphatidylcholine hydroperoxide were strongly inhibited by iodoacetate. After correction for the activity toward cumene hydroperoxide, it was shown that cumene hydroperoxide is mainly reduced by cytosolic glutathione peroxidase. Although the specific activity was lower than that of cytosolic glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase showed activity toward not only phosphatidylcholine hydroperoxide but also phosphatidylethanolamine hydroperoxide. These results suggest that cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase share a role in the reduction of hydroperoxide, but non-Se-dependent glutathione peroxidase (glutathione S-transferase) plays a lesser role.

PubMedID: 18237028 Hyperhomocysteinemia is associated with a lot of diseases including cardiovascular diseases and neural tube defect, but it has not been clarified exactly which mechanism is responsible for occurence disease. Here, homocysteine (Hcy) and cysteine (Cys), which are thiol containing amino acids, were examined for their effect on glutathione peroxidase (GPx) activity. It was observed that the GPx-1 activity was inhibited under severe hyperhomocysteinemia (50-500 µM Hcy) conditions, especially at low glutathione concentrations but that cysteine increased GPx-1 activity at low glutathione concentrations and inhibition clearly appeared at 500 µM Cys concentration. 2003YL7 was supported by C¸ ukurova Foundation (2003YL7).

We studied the effect of mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on the antioxidant activity of the glutathione system and NADPH-generating enzymes in liver and blood serum of rats with hyperglycemia induced by protamine sulfate. It was found that intraperitoneal injection of SkQ1 prevented both decrease in reduced glutathione level and increase in activity of glutathione system enzymes--glutathione peroxidase, glutathione reductase, and glutathione transferase. Activity of NADPH-generating enzymes--glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase--was also attenuated by SkQ1. Probably, in this model of hyperglycemia, decreased level of reactive oxygen species in mitochondria led to the decreased burden on the glutathione antioxidant system and NADPH-generating enzymes. Thus, SkQ1 appears to be a promising compound for the treatment and/or prevention of the adverse effects of hyperglycemia.

SUMMARYPhospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a major antioxidant enzyme, which plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We isolated and characterized a full-length cDNA sequence encodingGPxgene from a blood fluke,Schistosoma japonicum(designatedSjGPx), which contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3′-untranslated region. Protein encoded bySjGPxdemonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Semi-quantitative reverse transcription PCR and Western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference approach was employed to investigate the effects of knockdown ofSjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. Significantly reduction in GPx enzyme activities, as well as obvious changes in morphology of intrauterine eggs followed the reduction inSjGPxtranscript level. We observed a 63·04% reduction in GPx activity and the eggs severely deformed. Our results revealed that SjGPx protein might be involved in the provision of enzyme activity during egg production.

Menadione (MD), a simplest synthetic quinone used with reactive oxygen species (ROSs) generating agents, causes damages to Saccharomyces cerevisiae KNU5377. To understand antioxidant defense mechanisms under adaptive stress response, we have performed biochemical analyses and the genome-wide gene expression analysis after exposure to 120 μM of MD with and without adaptation. KNU5377 could survive at higher concentration of MD, 680 μM for 1 h, comparing with S. cerevisiae W303, ATCC24858 as reference strains. Glutathione peroxidase (GPX2), thioredoxin peroxidase 2 (TSA2), and thioredoxin reductase (TRR1) were up-regulated over 10-fold after treatment of 120 μM. Superoxide dismutase (SOD1), catalase (CTA1), glucose-6-phosphate dehydrogenase (ZWF1), glutathione peroxidase (GPX2, HYR1), glutathione reductase (GLR1), thioredoxin peroxidase (AHP1), and thioredoxin 2 (TRX2) were also strongly induced after adaptation. Additionally, transcriptional analysis of those genes was well corresponded with up-regulated protein expression and enzyme activity. Antioxidant defense mechanisms containing SOD1, ZWF and TRX2 maybe orchestrate by the different levels at the same time and function to produce effective defense systems. These results suggest that specific mechanisms including the three proteins were well organized for acquiring adaptive response to MD in this strain.

Glutathione peroxidase (GPx) is one of the most important antioxidative selenoenzymes in living organisms. The novel GPx mimic 6,6′-ditellurobis(6-deoxy-β-cyclodextrin) (6-TeCD) was prepared and evaluated for its capacity to catalyze the reduction of H2O2, tert-butyl hydroperoxide (t-BuOOH), and cumene hydroperoxide (CuOOH) by glutathione (GSH) or 3-carboxy-4-nitrobenzenethiol (ArSH). Compared the ArSH assay with the coupled reductase assay, we found that 6-TeCD exhibited strong substrate specificity for aromatic thiol substrate. The specificity led to efficient peroxidase activity almost 100,000-fold than that for a well-known GPx mimic diphenyl diselenide (PhSeSePh). Furthermore, reduction of lipophilic CuOOH was proceeded ca. 30 times faster than the more hydrophilic H2O2, which cannot bind into the hydrophobic cavity of β-cyclodextrin. Thus, it seemed that catalytic activity of cyclodextrin-derived GPx models strongly depends on the structurally different both substrates hydroperoxides (ROOH) and thiols.

In an alternate reading frame overlapping the viral envelope gene, HIV-1 has been shown to encoded a truncated glutathione peroxidase (GPx) module. Essential active site residues of the catalytic core regions of mammalian GPx sequences are conserved in the putative viral GPx (vGPx, encoded by the env-fs gene). Cells transfected with an HIV-1 env-fs construct show up to a 100% increase in GPx enzyme activity, and are protected against the loss of mitochondrial transmembrane potential and subsequent cell death induced by exogenous oxidants or mitochondrial reactive oxygen species. An intact vGPx gene was observed to be more common in HIV-1-infected long-term non-progressors, as compared to HIV-1 isolates from patients developing AIDS. An antioxidant/antiapoptotic protective role of the vGPx is also consistent with the observation that -1 frameshifting induced by the HIV-1 env-fs sequence AAAAAGA (which contains a potential "hungry" arginine codon, AGA) increases during arginine deficiency, which has been associated with increased oxidative stress. Under arginine-limited conditions, nitric oxide synthase generates superoxide, which rapidly combines with NO to form peroxynitrite, which can cause activated T-cells to undergo apoptosis. Thus, biosynthesis of the HIV-1 GPx as an adaptive response to low arginine conditions might delay oxidant-induced apoptotic cell death, providing an enhanced opportunity for viral replication.

Glutathione peroxidases catalyse the reduction of peroxides by reduced glutathione. To determine if these enzymes are important for resistance to oxidative stress and evasion of the innate immune system by the fungal pathogen Cryptococcus neoformans, two glutathione peroxidase homologues, which share 38 % identity, were identified and investigated. In this study, these peroxidases, Gpx1 and Gpx2, their localization, their contribution to total glutathione peroxidase activity, and their importance to the oxidative and nitrosative stress resistance of C. neoformans are described. It is shown that the two glutathione peroxidase genes are differentially expressed in response to stress. While both GPX1 and GPX2 are induced during t-butylhydroperoxide or cumene hydroperoxide stress and repressed during nitric oxide stress, only GPX2 is induced in response to hydrogen peroxide stress. Deletion mutants of each and both of the glutathione peroxidases were generated, and it was found that they are sensitive to various peroxide stresses while showing wild-type resistance to other oxidant stresses, such as superoxide and nitric oxide. While the glutathione peroxidase mutants are slightly sensitive to oxidant killing by macrophages, they exhibit wild-type virulence in a mouse model of cryptococcosis.