Volume 384, Issue 4

The antioxidant glutathione (GSH) is essential for the cellular detoxification of reactive oxygen species in brain cells. A compromised GSH system in the brain has been connected with the oxidative stress occuring in neurological diseases. Recent data demonstrate that besides intracellular functions GSH has also important extracellular functions in brain. In this respect astrocytes appear to play a key role in the GSH metabolism of the brain, since astroglial GSH export is essential for providing GSH precursors to neurons. Of the different brain cell types studied in vitro only astrocytes release substantial amounts of GSH. In addition, during oxidative stress astrocytes efficiently export glutathione disulfide (GSSG). The multidrug resistance protein 1 participates in both the export of GSH and GSSG from astrocytes. This review focuses on recent results on the export of GSH and GSSG from brain cells as well as on the functions of extracellular GSH in the brain. In addition, implications of disturbed GSH pathways in brain for neurodegenerative diseases will be discussed.

Reactive oxygen species are the unwanted by-products of aerobic metabolism. To protect cells against their potentially lethal effects a series of pathways have evolved that are collectively called the oxidative defence system. In most eukaryotes, catalases and selenium-dependent glutathione peroxidases form the front line of defence against hydroperoxide-mediated damage. However, these activities are lacking in members of the Trypanosomatidae family of protozoan parasites. Instead these organisms contain several enzyme-mediated pathways for removal of hydroperoxides that are centred upon the unusual thiol trypanothione. Here we discuss the biochemical properties of one group of these enzymes, the non-selenium glutathione-dependent peroxidases, and outline the roles that they play in protecting the parasite against hydroperoxides associated with biological membranes.

Adaptation to oxidative and nitrosative stress occurs in cells first exposed to a nontoxic stress, resulting in the ability to tolerate a toxic challenge of the same or a related oxidant. Adaptation is observed in a wide variety of cells including endothelial cells on exposure to nitric oxide or oxidized lipids, and lung epithelial cells exposed to air-borne pollutants and toxicants. This acquired characteristic has been related to the regulation of a family of stress responding proteins including those that control the synthesis of the intracellular antioxidant glutathione. The focus of this article, which includes a review of recent results along with new data, is the regulation and signaling of glutathione biosynthesis, especially those relating to adaptive mechanisms. These concepts are illustrated with examples using nitric oxide and oxidized low density lipoprotein mediated adaptation to oxidative stress. These data are discussed in the context of other adaptive mechanisms relating to glutathione synthesis including those from dietary constituents such as curcumin.

The bis(glutathionyl)spermidine trypanothione exclusively occurs in parasitic protozoa of the order Kinetoplastida, such as trypanosomes and leishmania, some of which are the causative agents of several tropical diseases. The dithiol is kept reduced by the flavoenzyme trypanothione reductase and the trypanothione system replaces in these parasites the nearly ubiquitous glutathione/glutathione reductase couple. Trypanothione is a reductant of thioredoxin and tryparedoxin, small dithiol proteins, which in turn deliver reducing equivalents for the synthesis of deoxyribonucleotides as well as for the detoxification of hydroperoxides by different peroxidases. Depending on the individual organism and the developmental state, the parasites also contain significant amounts of glutathione, mono-glutathionylspermidine and ovothiol, whereby all four low molecular mass thiols are directly (trypanothione and monoglutathionylspermidine) or indirectly (glutathione and ovothiol) maintained in the reduced state by trypanothione reductase. Thus the trypanothione system is central for any thiol regeneration and trypanothione reductase has been shown to be an essential enzyme in these parasites. The absence of this pathway from the mammalian host and the sensitivity of trypanosomatids toward oxidative stress render the enzymes of the trypanothione metabolism attractive target molecules for the rational development of new drugs against African sleeping sickness, Chagas' disease and the different forms of leishmaniasis.

When present as a trophozoite in human erythrocytes, the malarial parasite Plasmodium falciparum exhibits an intense glutathione metabolism. Glutathione plays a role not only in antioxidative defense and in maintaining the reducing environment of the cytosol. Many of the known glutathione-dependent processes are directly related to the specific lifestyle of the parasite. Reduced glutathione (GSH) supports rapid cell growth by providing electrons for deoxyribonucleotide synthesis and it takes part in detoxifying heme, a product of hemoglobin digestion. Free radicals generated in the parasite can be scavenged in reaction sequences involving the thiyl radical GS as well as the thiolate GS. As a substrate of glutathione S-transferase, glutathione is conjugated to nondegradable compounds including antimalarial drugs. Furthermore, it is the coenzyme of the glyoxalase system which detoxifies methylglyoxal, a byproduct of the intense glycolysis taking place in the trophozoite. Proteins involved in GSH-dependent processes include glutathione reductase, glutaredoxins, glyoxalase I and II, glutathione S-transferases, and thioredoxins. These proteins, as well as the ATP-dependent enzymes of glutathione synthesis, are studied as factors in the pathophysiology of malaria but also as potential drug targets. Methylene blue, an inhibitor of the structurally known P. falciparum glutathione reductase, appears to be a promising antimalarial medication when given in combination with chloroquine.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When the cellular production of ROS exceeds the cell's antioxidant capacity, cellular macromolecules such as lipids, proteins and DNA can be damaged. Because of this, 'oxidative stress' is thought to contribute to aging and pathogenesis of a variety of human diseases. However, in the last 10-15 years, a considerable body of evidence has accumulated that ROS serve as subcellular messengers, and play a role in gene regulation and signal transduction pathways, which may be involved in defensive mechanisms against oxidative stress. This review focuses on oxidative stress caused by the inactivation of glutathione peroxidase (GPx), a major peroxide scavenging enzyme. GPx is inactivated by a variety of physiological substances, including nitric oxide and carbonyl compounds in vitro and in cell culture. Decreased GPx activity has also been reported in tissues where oxidative stress occurs in several pathological animal models. The accumulation of increased levels of peroxide resulting from inactivation of GPx may act as a second messenger and regulate expression of antiapoptotic genes and the GPx itself to protect against cell damage. These findings suggest that GPx undergoes inactivation under various conditions such as nitroxidative stress and glycoxidative stress, and that these changes are a common feature of various types of oxidative stress which may be associated with the modification of redox regulation and cellular function.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx; EC 1.11.1.12), a broad-spectrum thiol-dependent peroxidase, deserves renewed interest as a regulatory factor in various signaling cascades and as a structural protein in sperm cells. We present a first attempt to identify catalytic intermediates and derivatives of the selenoprotein by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/ESI-MS/MS) and to explain observed specificities by molecular modeling. The ground state enzyme E proved to correspond to position 3-170 of the deduced porcine sequence with selenium being present as selenocysteine at position 46. The selenenic acid form, which is considered to be the first catalytic intermediate F formed by reaction with hydroperoxide, could not be identified. The second catalytic intermediate G was detected as Se-glutathionylated enzyme. This intermediate is generated in the reverse reaction where the active site selenol interacts with glutathione disulfide (GSSG). According to molecular models, specific binding of reduced glutathione (GSH) and of GSSG is inter alia facilitated by electrostatic attraction of Lys-48 and Lys-125. Polymerization of PHGPx is obtained under oxidizing conditions in the absence of low molecular weight thiols. Analysis of MS spectra revealed that the process is due to a selective reaction of Sec-46 with Cys-148' resulting in linear polymers representing deadend intermediates (G'). FT Docking of PHGPx molecules allowed reactions of Sec-46 with either Cys-66', Cys-107', Cys-168' or Cys-148', the latter option being most likely as judged by the number of proposed intermediates with reasonable hydrogen bonds, interaction energies and interface areas. We conclude that the same catalytic principles, depending on the conditions, can drive the diverse actions of PHGPx, i.e. hydroperoxide reduction, GSSG reduction, S-derivatization and self-incorporation into biological structures.

ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsinlike activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IκBα half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or proreducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.

Selenium-dependent glutathione peroxidase isoenzymes-1 and -2 are the major glutathione-dependent H2O2-reducing activities in the epithelium of the mid to lower gastrointestinal tract. The two isoenzymes protect mice against ileocolitis. We have found that luminal microflora are required for colitis to develop in mice deficient in GPX-1 and GPX-2 activity (GPX-DKO). Within 7 days of association with microflora, previously asymptomatic germfree GPX-DKO mice developed severe acute colitis while their littermates with at least one wildtype Gpx1 or Gpx2 gene remained virtually symptomfree. Microflora also affected Gpx2 gene expression. Gpx2, but not Gpx1, mRNA levels were elevated 4-5 fold in the ileum and colon in conventionally reared or microflora-associated adult mice compared with germ-free mice. Since the gastrointestinal tract microflora undergo major changes 2-3 weeks after birth, from relatively benign to a potentially stressful composition, we examined postnatal Gpx2 gene expression. The jejunal and ileal GPX-2 activity levels were low in two to three weekold mice and increased 5-7 fold during the next two weeks. GPX-2 activity levels were correlated with the mRNA levels. Colon Gpx2 mRNA levels held steady at about 50% of adult levels from 12-21 days of age but were several times higher than ileal levels. Our results suggest that ileal Gpx2 mRNA and GPX-2 activity levels are induced by luminal microflora. This response is consistent with a role for GPX as an antiinflammatory activity.

Interleukin-1 signaling is initiated by recruitment of adapter proteins and kinases to the type I interleukin-1 receptor (IL-1RI). It is modulated by accompanying redox processes at various levels, such as (auto-) phosphorylation of the IL-1RIassociated kinase IRAK, the phosphorylation of IκB and translocation and transcriptional activity of NFκB. Here we demonstrate that the thiolmodifying agents diamide, menadione, and phenylarsine oxide (PAO) block the recruitment of IRAK to the receptor without inhibiting kinase activity in the immunoprecipitated IL-1RI complex in the human epithelial cell line ECV304 and the murine T cell line EL-4. Inhibition of IRAK receptor association by menadione is reversible in a GSH-dependent manner, while the PAO effect proved to be irreversible. Phospholipid hydroperoxide glutathione peroxidase attenuates inhibition by menadione. Recruitment correlates with the presence of thiol groups in IRAK that were available for IAIT-labeling. We conclude that recruitment of IRAK to the IL-1RI is redox regulated by the glutathione system, a reduced status being a prerequisite for an appropiate IL-1 response.

Tryparedoxin peroxidases (TXNPx) are peroxiredoxintype enzymes that detoxify hydroperoxides in trypanosomatids. Reduction equivalents are provided by trypanothione [T(SH)2] via tryparedoxin (TXN). The T(SH)2-dependent peroxidase system was reconstituted from TXNPx and TXN of T. brucei brucei (TbTXNPx and TbTXN). TbTXNPx efficiently reduces organic hydroperoxides and is specifically reduced by TbTXN, less efficiently by thioredoxin, but not by glutathione (GSH) or T(SH)2. The kinetic pattern does not comply with a simple rate equation but suggests negative cooperativity of reaction centers. Gel permeation of oxidized TbTXNPx yields peaks corresponding to a decamer and higher aggregates. Electron microscopy shows regular ring structures in the decamer peak. Upon reduction, the rings tend to depolymerise forming openchain oligomers. Co-oxidation of TbTXNPx with TbTXNC43S yields a dead-end intermediate mimicking the catalytic intermediate. Its size complies with a stoichiometry of one TXN per subunit of TXNPx. Electron microscopy of the intermediate displays pentangular structures that are compatible with a model of a decameric TbTXNPx ring with ten bound TbTXN molecules. The redox-dependent changes in shape and aggregation state, the kinetic pattern and molecular models support the view that, upon oxidation of a reaction center, other subunits adopt a conformation that has lower reactivity with the hydroperoxide.

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) is present in at least three different isoforms in testis: as a cytosolic, as a mitochondrial, and as a nuclear protein. We have recently shown that a sperm nucleus-specific glutathione peroxidase (snGPx) is identical to the mitochondrial and cytosolic forms of PHGPx apart from its N-terminus. This arginine-rich N-terminus of snGPx, reminiscent of protamines, is encoded by an alternative exon located in the first intron of the PHGPx gene and is responsible for nuclear localisation and chromatin binding of snGPx [Pfeifer et al., FASEB J. 15 (2001), pp. 1236-1238]. By using a combination of techniques including selective cloning of mRNA 5' ends, RT-PCR, and S1 analyses, we provide evidence that the transcript encoding the nuclear form is generated by transcription initiation at an alternative promoter and not by alternative splicing. We show that the major transcription start region is located at 12 to 14 upstream of the AUG translation initiation site of the sperm nucleus-specific exon and lacks a TATA box. Two minor TATA-less transcription initiation sites are located at around -30 and -45. We have shown by in situ hybridisation that snGPx expression in testis, like protamine expression, is restricted to late stages of spermatogenesis whereas PHGPx expression is only found in spermatocytes and early spermatids. These findings have to be taken into account when studying either the differential regulation of PHGPx and snGPx expression in testis or the impact of putative mutations in snGPx on male fertility in man.

Exogenous and endogenous agents including products generated by oxidative stress, chemotherapeutics and bacterial lipids, activate multiple cellular signaling pathways, resulting either in mitogenesis or in apoptosis. Glutathione transferases (GSTs) appear not only to be prominent catalysts of detoxication reactions, but also to play a pivotal role in signaling by interacting with multiple proteins in pathways induced by cellular stress. Using two peptide libraries (a 9-mer and a 15-mer) displayed on phage, novel GST-peptide interactions were identified using human GST A1-1, GST P1-1 and GST M2-2 as targets. The isolated peptides have high sequence similarity with proteins such as TRAF4-associated factor 1, G protein-coupled receptor MRGX3, tumor necrosis factor superfamily (member 9), and c-Jun N-terminal kinase 3.

Trypanothione [T(SH)2], the major redox mediator in pathogenic trypanosomatids, is synthetized stepwise by two distinct enzymes in Crithidia fasciculata, while in Trypanosoma cruzi a single enzyme catalyzes both steps. A fulllength reading frame presumed to encode trypanothione synthetase (TryS) was obtained by PCR using DNA of T. brucei as template and primers based on fragments of putative TryS genes. The recombinant protein produced by E. coli Origami (DE3) was purified to homogeneity by chelate and ion exchange chromatography. The enzyme catalyzed both reactions of T(SH)2 biosynthesis. Thus, T(SH)2 synthesis appears to be similar in African (T. brucei) and New World (T. cruzi) trypanosomes but distinct from that of Crithidia.

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanylaminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAPspecific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The nondiscriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.

Activating transcription factor 2 (ATF2) belongs to the family of b asic region leucine zip per (bZIP) proteins that are characterized by the presence of a basic domain that functions as the DNAbinding domain and a leucine zipper domain that is required for dimerization. Together with bZIP proteins of the Fos and Jun families, ATF2 constitutes the AP-1 transcription factor complex. The biological activity of ATF2 is controlled by phosphorylation of two threonine residues within the N-terminal activation domain. Unphosphorylated ATF2 is trancriptionally silent, excluding simple overexpression studies to identify transcriptional targets of ATF2. We therefore decided to construct a constitutively active ATF2 mutant that would allow us to uncouple the investigation of transcriptional targets and biological functions of ATF2 from the variety of signaling pathways that lead to an activation of ATF2. We exchanged the phosphorylationdependent activation domain of ATF2 with the constitutively active transcriptional activation domain of the transcription factor CREB2. In transient transfection experiments, this constitutively active ATF2 mutant stimulated c-jun, tumor necrosis factor α, and Fas ligand promoter activities. The transcriptional activity of the constitutively active ATF2 mutant could be impaired by dominantnegative forms of ATF2 or c-jun, indicating that ATF2 and c-jun utilize a similar dimerization code. In contrast, a dominantnegative CREB2 mutant did not impair ATF2-mediated transcriptional activation, suggesting that CREB2 exhibits a different dimerization specificity than ATF2 or c-jun.

The pyruvate dehydrogenase from Escherichia coli showed a primary kinetic isotope effect when its overall reaction or the partial reaction of the pyruvate dehydrogenase component were tested in deuterium oxide. The Michaelis constants for pyruvate were nearly unchanged, but the maximum velocities in water and deuterium oxide differed, their ratio being DV = 1.7 for the overall reaction and DV = 2.1 for the E1p reaction. The pH profile and, accordingly, the δpK1 and δpK2 values were shifted by 0.6 units to higher pL values. A linear proton inventory curve was obtained when varying the atom fractions of protons relative to deuterons from 100 to 0%. This is an indication for a single proton transfer. It is proposed that this relatively weak primary isotope effect may be caused by the protonation of the N1 nitrogen at the pyrimidine ring of the cofactor by an adjacent glutamate residue. The proton of its carboxylic group exchanges very fast with deuterons of the solvent.

Liver heme oxygenase (HO) activity is higher in selenium-deficient rats than in control animals under basal conditions and is further increased in them, but not in controls, by phenobarbital treatment. In the present study we characterized liver HO induction by selenium deficiency using molecular methods. Severe selenium deficiency in rats caused a doubling of liver HO activity without affecting spleen, kidney, brain, or testis HO activities. HO-1 protein and mRNA were increased to accompany the increased HO activity, but HO-2 protein and mRNA were not increased. Fractionation of the liver into hepatocyte and Kupffer cell/endothelial cell fractions revealed that the increased HO activity resides in the hepatocyte fraction. Immunohistochemical localization of HO-1 protein confirms the induction of HO-1 taking place solely in hepatocytes and throughout the liver lobule. Phenobarbital treatment sharply increased HO-1 mRNA and protein expression in selenium-deficient liver and HO activity in hepatocytes, but had no effect in control liver or in the Kupffer cell/endothelial cell fraction of selenium-deficient liver. Electrophoretic mobility shift assays showed increased AP-1 binding activity, suggesting an involvement of this redoxsensitive transcription factor in the induction by phenobarbital of HO-1 in selenium deficiency. We speculate that selenium deficiency affects hepatic antioxidant selenoproteins, resulting in an upregulation of HO-1.