Glutathione Peroxidase-Like Research Articles

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.

Transcriptomic response of intertidal brittle star Ophiothrix exigua to seasonal variation.

Adaptation to seasonal change is essential for survival, and is especially critical for organisms living in physically harsh environments. Brittle stars (Ophiothrix), known as a keystone species, inhabiting the intertidal rocky ecosystem are affected by multiple stressors, but molecular insights into their adaptation remain poorly studied. In the present study, transcriptomic responses of Ophiothrix exigua from the intertidal habitats of the North Pacific Ocean during summer and winter are reported. A total of 12,844 differentially expressed genes (DEGs) were identified. Of these, 7102 genes were up-regulated and 5742 genes were down-regulated in summer relative to winter. One hundred fifty-two key DEGs, including 31 up-regulated and 121 down-regulated genes, were categorized into three major subcategories and seven subclasses. The key DEGs included heat shock cognate protein 70 (HSC70), toll-like receptor-2 (TLR2), cAMP-dependent protein kinase catalytic subunit beta-like isoform X2 (PKA), serine/threonine-protein kinase mTOR (MTOR), and ras-related c3 botulinum toxin substrate 1 isoform X1 (RAC1). Glutathione peroxidase-like (GPX) and tubulin superfamily members (TUBA, TUBB) were consistent across seasons. The main defense-related pathways in brittle star were phagosome, apoptosis, and glutathione metabolism. These findings would greatly enhance our understanding of the genomic basis of environmental adaptation in intertidal invertebrates.

Crosstalk between the Arabidopsis Glutathione Peroxidase-Like 5 Isoenzyme (AtGPXL5) and Ethylene.

Glutathione peroxidases (GPXs) are important antioxidant enzymes in animals. Plants contain GPX-like (GPXL) enzymes, which—in contrast to GPXs—contain cysteine in their active site instead of selenocysteine. Although several studies proved their importance in development and stress responses, their interaction with ethylene (ET) signalling is not known. Our aim was to investigate the involvement of AtGPXL5 in ET biosynthesis and/or signalling using Atgpxl5 mutant and AtGPXL5 cDNA-overexpressing (OX-AtGPXL5) lines. Four-day-old dark-grown Atgpxl5 seedlings had shorter hypocotyls and primary roots, while OX-AtGPXL5 seedlings exhibited a similar phenotype as wild type under normal conditions. Six-week-old OX-AtGPXL5 plants contained less H2O2 and malondialdehyde, but higher polyamine and similar ascorbate- and glutathione contents and redox potential (EGSH) than the Col-0. One-day treatment with the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced the activity of glutathione- and thioredoxin peroxidases and some other ROS-processing enzymes. In the Atgpxl5 mutants, the EGSH became more oxidised; parallelly, it produced more ethylene after the ACC treatment than other genotypes. Although the enhanced ET evolution measured in the Atgpxl5 mutant can be the result of the increased ROS level, the altered expression pattern of ET-related genes both in the Atgpxl5 and OX-AtGPXL5 plants suggests the interplay between AtGPXL5 and ethylene signalling.

Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance.

This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)—both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))—and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14+/PI−) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, could be used as markers of sperm cryotolerance. Further research addressing the relationship of these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers is warranted.

(Z,Z)-Selanediylbis(2-propenamides): Novel Class of Organoselenium Compounds with High Glutathione Peroxidase-Like Activity. Regio- and Stereoselective Reaction of Sodium Selenide with 3-Trimethylsilyl-2-propynamides.

The efficient regio- and stereoselective synthesis of (Z,Z)-3,3′-selanediylbis(2-propenamides) in 76–93% yields was developed based on the reaction of sodium selenide with 3-trimethylsilyl-2-propynamides. (Z,Z)-3,3′-Selanediylbis(2-propenamides) are a novel class of organoselenium compounds. To date, not a single representative of 3,3′-selanediylbis(2-propenamides) has been described in the literature. Studying glutathione peroxidase-like properties by a model reaction showed that the activity of the obtained products significantly varies depending on the organic moieties in the amide group. Divinyl selenide, which contains two lipophilic cyclohexyl substituents in the amide group, exhibits very high glutathione peroxidase-like activity and this compound is considerably superior to other products in this respect.

Overexpression of the Arabidopsis glutathione peroxidase-like 5 gene (AtGPXL5) resulted in altered plant development and redox status

Plant’s glutathione peroxidase-like (GPXL) enzymes are thiol-based peroxidases catalysing the reduction of H2O2 or hydroperoxides to water or alcohols using reduced glutathione (GSH) or thioredoxin as an electron donor. Arabidopsis thaliana possess eight isoenzymes having different roles in redox-dependent processes. AtGPXL5 is a poorly known plasma membrane associated enzyme. We have investigated the role of AtGPXL5 in development and responses to salt using AtGPXL5-overexpressing lines (OX-AtGPXL5) and Atgpxl5 mutants. Constitutive overexpression of AtGPXL5 increased the transcription of the gene by 17–24 times in 6-week-old plants. In OX-AtGPXL5 plants, the activity of glutathione peroxidase, thioredoxin peroxidase and most of the main antioxidant enzymes were like in the wild type Col-0, but the amount of GSH was increased, thus the redox potential became more negative compared to the wild type. The well-preserved germination rate, seedling growth and chlorophyll content of the OX-AtGPXL5 seedlings in the presence of 100 mM NaCl indicated the increased salt tolerance of AtGPXL5-overexpressing plants. In agreement, the Atgpxl5 knockdown mutants had enhanced salt stress sensitivity in comparison to the wild type. Our results indicate that AtGPXL5 may have function in the fine-tuning of ROS levels and redox status during salt stress.

Comprehensive analysis of antioxidant mechanisms in Arabidopsis glutathione peroxidase-like mutants under salt- and osmotic stress reveals organ-specific significance of the AtGPXL’s activities

Plant glutathione peroxidases contain cysteine in their active site instead of selenocysteine, and most of them use the thioredoxin (TRX) system more efficiently than the glutathione (GSH) system during the reduction of H2O2 and lipid peroxides. Recently, the more precise glutathione peroxidase-like (GPXL) name was adopted for the eight Arabidopsis thaliana isoenzymes. In this paper we have compared the effect of osmotic and salt stresses on the 6-week-old T-DNA insertion mutants (Atgpxl1-8) grown hydroponically. The glutathione peroxidase (GPOX) activity measured with cumene hydroperoxide substrate in the wild type Arabidopsis shoots and roots was 2–5 times higher than the thioredoxin peroxidase (TPOX) activity. Mutation in one of the eight AtGPXLs resulted in decreased TPOX activity in untreated shoots and, in contrary to the wild type, this activity did not increase under stress, verifying the connection with TRX system. The level of reduced ascorbate significantly altered in shoots and the amount of GSH in roots under both control conditions and after 2 days of stress treatments. While positive correlations were found between GSH and TPOX activity in the wild type shoots and roots, the connection between the AtGPXLs and the GSH pool was stronger in roots than in shoots. Nevertheless, the TPOX activity increased in Atgpxls roots when the GSH content decreased, indicating the relationship between the GSH and TRX systems. The AtGPXL expression in mutant plants showed that some isoenzymes are regulated jointly. Furthermore, while AtGPXLs were generally down-regulated, several stress-inducible transcription factor genes were up-regulated, especially after applying osmotic stress.

Glutathione Peroxidase-Like Activity of Amino-Substituted Water-Soluble Cyclic Selenides: A Shift of the Major Catalytic Cycle in Methanol.

We previously reported that water-soluble cyclic selenides can mimic the antioxidative function of glutathione peroxidase (GPx) in water through a simple catalytic cycle, in which the selenide (>Se) is oxidized by H2O2 to the selenoxide (>Se=O) and the selenoxide is reduced by a thiol back to the selenide. In methanol, however, the GPx-like activity could not be explained by this simple scenario. To look into the reasons for the unusual behaviors in methanol, monoamino-substituted cyclic selenides with a variable ring size were synthesized, and the intermediates of the catalytic cycle were characterized by means of 77Se-NMR and LC–MS spectroscopies. In water, it was confirmed that the selenide and the selenoxide mainly contribute to the antioxidative function, though a slight contribution from the dihydroxy selenane (>Se(OH)2) was also suggested. In methanol, on the other hand, other active species, such as hydroxyselenonium (>Se+–OH) and hydroxy perhydroxy selenane (>Se(OH)(OOH)), could be generated to build another catalytic cycle. This over-oxidation would be more feasible for amino-substituted cyclic selenides, probably because the ammonium (NH3+) group would transfer a proton to the selenoxide moiety to produce a hydroxyselenonium species in the absence of an additional proton source. Thus, a shift of the major catalytic cycle in methanol would make the GPx-like antioxidative function of selenides perplexing.

Selenium/Tellurium-Containing Hyperbranched Polymers: Effect of Molecular Weight and Degree of Branching on Glutathione Peroxidase-Like Activity

A series of novel hyperbranched polyselenides and polytellurides with multiple catalytic sites at the branching units has been synthesized via the polycondensation of A2 + B3 monomers. The GPx-like activities of these polymer mimics were assessed and it was found that the polytellurides showed higher GPx-like activities than the corresponding polyselenides. Interestingly, the polymers with higher molecular weights and degree of branching (DB) showed higher GPx-like activities than the analogous lower molecular weight polymer. The enhancement in the catalytical activity of the hyperbranched polymers with increasing molecular weight affirmed the importance of the incorporation of multiple catalytic groups in the macromolecule which increases the local concentration of catalytic sites.

Synthesis of Cyclic Selenenate/Seleninate Esters Stabilized byortho‐Nitro Coordination: Their Glutathione Peroxidase‐Like Activities

The syntheses of selenenate/seleninate esters and related derivatives by aromatic nucleophilic substitution (S(N)Ar) reactions of 2-bromo-3-nitrobenzylalcohol (13) and 2-bromo-3-nitrobenzaldehyde (17) with Na(2)Se(2)/nBuSeNa are described. The reaction of 13 with Na(2)Se(2) at room temperature afforded 7-nitro-1,2-benzisoselenole(3H) (15) instead of the desired diaryl diselenide 14. Oxidation of selenenate ester 15 with hydrogen peroxide afforded the corresponding selenium(IV) derivative, 7-nitro-1,2-benzisoselenole(3H) selenium oxide (18). 2-(Butylselanyl)-3-nitrobenzaldehyde (19) was synthesized by treating compound 17 with in situ generated nBuSeNa. The bromination reaction of selenide 19 did not afford the expected arylselenenyl bromide 20, instead, it resulted in the formation of the unexpected 7-nitro-1,2-benzisoselenol(3H)-3-ol (21) and 3,3'-oxybis(7-nitro-1,2-benzisoselenole(3H)) (22), respectively. The facile formation of heterocycles 21 and 22 is rationalized in terms of the aromatic ring strain in selenenyl bromide 20. The presence of intramolecular secondary Se···O interactions in esters 15, 18, 21, 22, and selenenic anhydride 29 has been confirmed by single-crystal X-ray diffraction studies as well as computational studies. The presence of an intramolecular Se···O interaction in esters 4b, 8, 15, 18, 21, and 22 has been further proved by natural bond orbital (NBO) and atoms in molecules (AIM) calculations. Glutathione peroxidase-like (GPx) antioxidant activities of 15, 18, 21, 22, and related heterocycles such as 7-nitro-1,2-benzisoselenol(2H)-3-one selenium oxide (4b), 7-nitro-1,2-benzisoselenol(2H)-3-one (8), and 29 have been determined by the coupled reductase assay.

Effect of an antioxidant, ebselen, on development of chronic cerebral vasospasm after subarachnoid hemorrhage in primates

BACKGROUND Oxidation and/or free radical reactions after subarachnoid hemorrhage (SAH) may be involved in the development of chronic cerebral vasospasm. The inhibition of these reactions is thought to be one of the therapeutic strategies for prevention of cerebral vasospasm. We investigated the effect of Ebselen, a synthetic seleno-organic compound, which exhibits anti-oxidation by glutathione peroxidaselike activity to inhibit free radical reactions by lipid peroxidation on the development of chronic cerebral vasospasm in a primate model. METHODS Seventeen monkeys were used. SAH was produced by introduction of a blood clot around the right middle cerebral artery and the right side of the circle of Willis in all animals. The monkeys were randomly divided into three groups according to Ebselen dosage: 1) no dosage or non-treated group; 2) high-dose Ebselen group; and 3) low-dose Ebselen group. The drug was administered at 10 mg/Kg in the high-dose group and 5 mg/Kg in the low-dose group twice a day in each group for 7 days after SAH. The vessel diameter was evaluated on angiograms before the induction of SAH and at Day 7 following SAH. RESULTS In the untreated group, the angiograms showed significant ( p < 0.05) reductions of the mean vessel caliber of the right internal carotid (ICA) (38 ± 10% reduction) and the middle cerebral artery (MCA) (56 ± 9.7%) compared with the baseline value before SAH. In the high-dose Ebselen-treated group, the mean percent reduction in vessel caliber of the right ICA (16 ± 11%) and MCA (28 ± 9.5%) on Day 7 angiograms were significantly ( p < 0.05) lower than those in the nontreated group, whereas the mean percent reduction of these vessels in the low-dose Ebselen-treated group showed no significant difference compared with the untreated group. CONCLUSIONS Chronic cerebral vasospasm was inhibited in the animals in which a relatively large amount of Ebselen was administered for 7 days after SAH. The results suggest that the oxidation or free radical reaction by lipid peroxidation after SAH might be involved in the pathogenesis of vasospasm, and that inhibition of these reactions by drugs, such as Ebselen, may have a promising effect for prevention of vasospasm.

Glutathione Peroxidase-Like Catalytic Activities of Ion-Exchange Resins Modified with Metalloporphyrins

Some of the ion-exchange resins modified with metalloporphyrins have been found to exhibit glutathione-peroxidase (GSHPx)-like catalytic activities for hydrogen peroxide and cumene hydroperoxide in the oxidative reaction of glu-tathione to disulfide. In particular, the anion-exchange resin modified with Mn(III)-tetrakis(sulfophenyl)porphine (Mn-TSPPr) exhibited stronger activity than GSHPx, and hence Mn-TSPPr was expected to be a good mimesis in place of GSHPx or immobilized GSHPx for the determination of peroxide.

Glutathione Peroxidase-Like Activity of Simple Selenium Compounds. Peroxides and the Heterocyclic N-Oxide Resazurin Acting as O-Atom Donors.

Selenite and selenocystamine [(CyaSe)2] efficiently activate the decomposition of H2O2 y GSH and by other thiols, as demonstrated using a leuco crystal violet POD-based H2O2 assay which is applicable (unlike other assays) also in presence of thiols. The GPx-like activities were estimated to be 3.6 and 2.7 μmol H2O2/min per μmol SeO3 2- and (CyaSe)2, respectively. Both selenium compounds also activate reduction of the heterocyclic N-oxide resazurin (RN→O) to resorufin (RN) by GSH; H2O2 competes with reduction of this dye. GSSeH and CyaSeH, formed by interaction of GSH with SeO3 2- and (CyaSe)2, respectively, are likely to be the active reductants. CyaSeH, generated γ-radiolytically from (CyaSe)2, exhibits an absorption peak at 243 nm and is removed by H2O2 with a rate constant of 9.7x102 ᴍ-1 s-1, and slightly slower by hydroperoxides. We have no evidence for one-electron interactions between GSSeH or CyaSeH and H2O2, with formation of free radical intermediates, as previously proposed in the case of selenium-activated reduction of cytochrome c by GSH (Levander et al., Biochemistry 23, 4591-4595 (1973)). Our results can be explained by O-atom transfer from the substrate to the active selenol group. RSeH + H2O2 (RN→O)→RSeOH + H2O (RN), and recycling of RSeOH to RSeH (+ H2O) by GSH, analogous to the selenenic acid pathway of GPx. The substrate specificity appears to be different, however, in that GPx is unable to catalyse RN→O reduction, and GSSeH hardly catalyses the decomposition of cumene- or t-butyl-hydroperoxide; CyaSeH, on the other hand, is active also with the hydroperoxides. RN→O is reduced to RN also by certain oxidizing free radicals, e.g. by the thiyl CyaS·; O -atom transfer may in this case lead to the generation of reactive oxyl radicals.