Mammalian Glutathione Peroxidase Research Articles

Oxidative stress drives divergent evolution of the glutathione peroxidase (GPX) gene family in mammals.

The molecular basis for adaptations to extreme environments can now be understood by interrogating the ever-increasing number of sequenced genomes. Mammals such as cetaceans, bats, and highland species can protect themselves from oxidative stress, a disruption in the balance of reactive oxygen species, which results in oxidative injury and cell damage. Here, we consider the evolution of the glutathione peroxidase (GPX) family of antioxidant enzymes by interrogating publicly available genome data from 70 mammalian species from all major clades. We identified 8 GPX subclasses ubiquitous to all mammalian groups. Mammalian GPX gene families resolved into the GPX4/7/8 and GPX1/2/3/5/6 groups and are characterized by several instances of gene duplication and loss, indicating a dynamic process of gene birth and death in mammals. Seven of the eight GPX subfamilies (all but GPX7) were under positive selection, with the residues under selection located at or close to active sites or at the dimer interface. We also reveal evidence of a correlation between ecological niches (e.g. high oxidative stress) and the divergent selection and gene copy number of GPX subclasses. Notably, a convergent expansion of GPX1 was observed in several independent lineages of mammals under oxidative stress and may be important for avoiding oxidative damage. Collectively, this study suggests that the GPX gene family has shaped the adaption of mammals to stressful environments.

Efficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli.

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. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNAUTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

Crystal and solution structural studies of mouse phospholipid hydroperoxide glutathione peroxidase 4.

The mammalian glutathione peroxidase (GPx) family is a key component of the cellular antioxidative defence system. Within this family, GPx4 has unique features as it accepts a large class of hydroperoxy lipid substrates and has a plethora of biological functions, including sperm maturation, regulation of apoptosis and cerebral embryogenesis. In this paper, the structure of the cytoplasmic isoform of mouse phospholipid hydroperoxide glutathione peroxidase (O70325-2 GPx4) with selenocysteine 46 mutated to cysteine is reported solved at 1.8 Å resolution using X-ray crystallography. Furthermore, solution data of an isotope-labelled GPx protein are presented.

Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration

The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4−/− mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4−/− livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4−/− pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4−/− mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.

Understanding mammalian glutathione peroxidase 7 in the light of its homologs

The glutathione peroxidase homologs (GPxs) efficiently reduce hydroperoxides using electrons from glutathione (GSH), thioredoxin (Trx), or protein disulfide isomerase (PDI). Trx is preferentially used by the GPxs of the majority of bacteria, invertebrates, plants, and fungi. GSH or PDI, instead, is preferentially used by vertebrate GPxs that operate by Sec or Cys catalysis, respectively. Mammalian GPx7 and GPx8 are unique homologs that contain a peroxidatic Cys (CP). Being reduced by PDI and located within the endoplasmic reticulum (ER), these enzymes have been involved in oxidative protein folding. Kinetic analysis indicates that oxidation of PDI by recombinant GPx7 occurs at a much faster rate than that of GSH. Nonetheless, activity measurement suggests that, at physiological concentrations, a competition between these two substrates takes place, with the rate of PDI oxidation by GPx7 controlled by the concentration of GSH, whereas the GSSG produced in the competing reaction contributes to the ER redox buffer. A mechanism has been proposed for GPx7 involving two Cys residues, in which an intramolecular disulfide of the CP is formed with an alleged resolving Cys (CR) located in the strongly conserved FPCNQ motif (C86 in humans), a noncanonical position in GPxs. Kinetic measurements and comparison with the other thiol peroxidases containing a functional CR suggest that a resolving function of C86 in the catalytic cycle is very unlikely. We propose that GPx7 is catalytically active as a 1-Cys-GPx, in which CP both reduces H2O2 and oxidizes PDI, and that the CP-C86 disulfide has instead the role of stabilizing the oxidized peroxidase in the absence of the reducing substrate.

The Involvement of Arabidopsis Glutathione Peroxidase 8 in the Suppression of Oxidative Damage in the Nucleus and Cytosol

A family of eight genes with homology to mammalian glutathione peroxidase (GPX) isoenzymes, designated AtGPX1-AtGPX8, has been identified in Arabidopsis thaliana. In this study we demonstrated the functional analysis of Arabidopsis AtGPX8 with peroxidase activity toward H(2)O(2) and lipid hydroperoxides using thioredoxin as an electron donor. The transcript and protein levels of AtGPX8 in Arabidopsis were up-regulated coordinately in response to oxidative damage caused by high-light (HL) stress or treatment with paraquat (PQ). Furthermore, the knockout Arabidopsis mutants of AtGPX8 (KO-gpx8) exhibited increased sensitivity to oxidative damage caused by PQ treatment in root elongation compared with the wild-type plants. In contrast, transgenic lines overexpressing AtGPX8 (Ox-AtGPX8) were less sensitive to oxidative damage than the wild-type plants. The levels of oxidized proteins in the KO-gpx8 and Ox-AtGPX8 lines were enhanced and suppressed, respectively, compared with the wild-type plants under HL stress or PQ treatment. The fusion protein of AtGPX8 tagged with green fluorescent protein was localized in the cytosol and nucleus of onion epidermal cells. In addition, the AtGPX8 protein was detected in the cytosolic and nuclear fractions prepared from leaves of Arabidopsis plants using the AtGPX8 antibody. Oxidative DNA damage under treatment with PQ increased in the wild-type and KO-gpx8 plants, while it decreased in the OX-AtGPX8 plants. These results suggest that AtGPX8 plays an important role in the protection of cellular components including nuclear DNA against oxidative stress.

Physiological functions of GPx2 and its role in inflammation‐triggered carcinogenesis

Mammalian glutathione peroxidases (GPxs) are reviewed with emphasis on the role of the gastrointestinal GPx2 in tumorigenesis. GPx2 ranks high in the hierarchy of selenoproteins, corroborating its importance. Colocalization of GPx2 with the Wnt pathway in crypt bases of the intestine and its induction by Wnt signals point to a role in mucosal homeostasis, but GPx2 might also support tumor growth when increased by a dysregulated Wnt pathway. In contrast, the induction of GPx2 by Nrf2 activators and the upregulation of COX2 in cells with a GPx2 knockdown reveal inhibition of inflammation and suggest prevention of inflammation-mediated carcinogenesis. The Janus-faced role of GPx2 has been confirmed in a mouse model of inflammation-associated colon carcinogenesis (AOM/DSS), where GPx2 deletion increased inflammation and consequently tumor development, but decreased tumor size. The model further revealed a GPx2-independent decrease in tumor development by selenium (Se) and detrimental effects of the Nrf2-activator sulforaphane in moderate Se deficiency.

Glutathione Peroxidases at Work on Epididymal Spermatozoa: An Example of the Dual Effect of Reactive Oxygen Species on Mammalian Male Fertilizing Ability

The mammalian glutathione peroxidase (GPx) gene family encodes bifunctional enzymes that can work either as classical reactive oxygen species (ROS) scavengers or as thiol peroxidases, thereby introducing disulfide bridges in thiol-containing proteins. These dual effects are nowhere better demonstrated than in epididymal maturing spermatozoa, where the concomitant actions of several GPx ensure the achievement of the structural maturation of sperm cells as well as their protection against ROS-induced damage. We review here the roles played by the sperm-associated forms of GPx4 (mitochondrial GPx4 and nuclear GPx4), the secreted GPx5 protein, and the epithelial proteins GPx1, GPx3, and cellular GPx4, all functioning in the mammalian epididymis at different stages of the sperm's epididymal journey, and in different epididymis compartments.

Kinetics and redox regulation of Gpx1, an atypical 2-Cys peroxiredoxin, in Saccharomyces cerevisiae

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.

Mammalian glutathione peroxidases control acquisition and maintenance of spermatozoa integrity 1

In mammals, posttesticular epididymal sperm maturation is considered an essential step in the transformation of immature testicular gametes to mature spermatozoa capable of fertilization. Reactive oxygen species (ROS) have been shown to be key actors in this maturation process, and it is now clear that ROS are central for sperm physiology in processes such as sperm maturation and capacitation. However, during epididymal maturation and storage and until the onset of fertilization, oxidative damage is a threat spermatozoa must face more than any other cells. Spermatozoa were found to be extremely sensitive to oxidative attacks correlated with lipid peroxidation, DNA damage, and impaired sperm motility, all affecting fertilization. To control the quantity of H(2)O(2) in the vicinity of male gametes, mammalian epididymis uses a panel of nonenzymatic and enzymatic scavengers, among which the glutathione peroxidase (GPx) family is largely represented. Among the various GPx proteins expressed in the mammalian epididymis, GPx4 and GPx5 occupy unique positions and functions that are reviewed in this paper. This paper underlines the importance of the GPx protein family in determining the fertilizing potential of mammalian spermatozoa. This is particularly relevant in the field of mammalian fertility and infertility as well as in the development of assisted medical procreation technologies and male gamete preservation techniques that are extensively used in human and animal reproduction programs.

Functional studies of an HIV‐1 encoded glutathione peroxidase

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.

Does the Active Site of Mammalian Glutathione Peroxidase (GPx) Contain Water Molecules? An ONIOM Study

In the present theoretical study, the active site of mammalian glutathione peroxidase (GPx) has been refined by including protein/active site interactions using the two-layer ONIOM(QM:MM) method. In this study, a full model consisting of 3113 atoms (with 86 active site atoms in the quantum mechanical (QM) region and the rest in the molecular mechanics (MM) region) has been constructed on the basis of the 2.9 A resolution X-ray structure of mammalian GPx. The “active site only” models give much larger root-mean-square (rms) deviations from the X-ray structure than the full model, indicating the importance of the protein environment on the structure of the active site. The still substantial rms errors of the optimized structures can be improved only when the full model complemented with two water molecules is considered, clearly indicating the existence of two missing water molecules at the active site of the mammalian GPx, which could be critical for the catalytic activity.

Molecular characterization of glutathione peroxidase-like protein in halotolerant Chlamydomonas sp. W80.

A cDNA clone encoding a glutathione peroxidase (GPX)-like protein was isolated from the cDNA library from halotolerant Chlamydomonas W80 (C. W80) by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 163 amino acids with a calculated molecular mass of 18 267 Da. No potential signal peptide was found. The deduced amino acid sequence of the cDNA showed 40-63% and 37-46% homology to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. The C. W80 GPX-like protein contained a normal cysteine residue instead of a selenocysteine at the catalytic site. However, it contained amino acid residues (glutamine and tryptophan) that are involved in three protein loops and are important for the catalytic activity in the mammalian GPX. Interestingly, the native and recombinant GPX-like proteins showed activities towards unsaturated fatty acid hydroperoxides, but not towards either H2O2 or phospholipid hydroperoxide. Transformed E. coli cells expressing the C. W80 GPX-like protein showed enhanced tolerance to 5% NaCl or 0.2 mM paraquat treatments. Accession number: The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number AB009083.

NADPH-dependent glutathione peroxidase-like proteins (Gpx-1, Gpx-2) reduce unsaturated fatty acid hydroperoxides in Synechocystis PCC 6803

Here we isolated and characterized two genes ( slr1171, slr1992) designated gpx-1 and gpx-2, respectively, encoding glutathione peroxidase (GPX)-like proteins (Gpx-1, Gpx-2) from Synechocystis PCC 6803. The deduced amino acid sequences for gpx-1 and gpx-2 showed high similarity to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. Surprisingly, both recombinant proteins in Escherichia coli were able to utilize NADPH, but not reduced glutathione, as an electron donor and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides as an acceptor. It seems accurate to refer to Gpx-1 and Gpx-2 as NADPH-dependent GPX-like proteins that serve as a new defense system for the reduction of unsaturated fatty acid hydroperoxides.

Nutrition, HIV, and drug abuse: the molecular basis of a unique role for selenium.

HIV-infected injection drug users (IDUs) often suffer from serious nutritional deficiencies. This is a concern because plasma levels of micronutrients such as vitamin B12, zinc, and selenium have been correlated with mortality risk in HIV-positive populations. Injection drug use also increases lipid peroxidation and other indicators of oxidative stress, which, combined with antioxidant deficiencies, can stimulate HIV-1 replication through activation of NF-kappaB transcription factors, while weakening immune defenses. As detailed herein, these prooxidant stimuli can also increase the pathogenic effects of HIV-1 by another mechanism, involving viral selenoproteins. Overlapping the envelope coding region, HIV-1 encodes a truncated glutathione peroxidase (GPx) gene (see #6 in reference list). Sequence analysis and molecular modeling show that this viral GPx (vGPx) module has highly significant structural similarity to known mammalian GPx, with conservation of the catalytic triad of selenocysteine (Sec), glutamine, and tryptophan. In addition to other functions, HIV-1 vGPx may serve as a negative regulator of proviral transcription, by acting as an NF-kappaB inhibitor (a known property of cellular GPx). Another potential selenoprotein coding function of HIV-1 is associated with the 3' end of the nef gene, which terminates in a conserved UGA (potential Sec) codon in the context of a sequence (Cys-Sec) identical to the C-terminal redox center of thioredoxin reductase, another cellular regulator of NF-kappaB. Thus, in combination with known cellular mechanisms involving Se, viral selenoproteins may represent a unique mechanism by which HIV-1 monitors and exploits an essential micronutrient to optimize its replication relative to the host.

Isolation and identification of a glutathione peroxidase homolog gene, gpxA, present in Neisseria meningitidis but absent in Neisseria gonorrhoeae

Antioxidant enzymes are thought to be important for the survival of pathogenic Neisseria species. We isolated a glutathione peroxidase-related gene (gpxA) from Neisseria meningitidis FAM20. The N. meningitidis glutathione peroxidase homolog was 49 to 57% identical to seven other glutathione peroxidase family members over a 49-amino-acid region which is conserved among various species. The gpxA sequence was present in all 7 meningococcal strains tested but absent in 10 gonococcal strains and 6 nonpathogenic neisserial strains as determined by Southern hybridization. The homology of gpxA to mammalian glutathione peroxidases and the presence of this gene specifically in the meningococcus suggest that it is important in the cellular metabolism or defense processes particular to this pathogen.

Identification of a glutathione peroxidase homolog in Neisseria meningitidis.

Glutathione peroxidase is an antioxidant enzyme found in a diverse array of eukaryotic species. We have determined the DNA sequence of a glutathione peroxidase homolog in the pathogenic bacterium Neisseria meningitidis. The sequence displays features of a functional gene, but lacks a selenocysteine-encoding in-frame TGA codon characteristic of most mammalian glutathione peroxidase genes. The derived amino acid sequence encoded by the N. meningitidis homolog predicts a 19.9 kDa protein that displays a high level of amino acid identity with other gluathione peroxidase sequences, particularly within four conserved regions of the enzyme.

A novel plant glutathione peroxidase-like protein provides tolerance to oxygen radicals generated by paraquat in Escherichia coli

Citrus salt-stress associated protein (Cit-SAP) reveals significant sequence homology to mammalian glutathione peroxidase (GP). In an attempt to assign biological function to this protein, transformed E. coli cells expressing Cit-SAP were examined for their ability to tolerate free radicals formed by paraquat, an O − 2 radical forming agent. In the presence of paraquat, the survival rate of the transformed bacteria expressing Cit-SAP was much higher as compared to the wild-type bacteria. The results support the assumption that Cit-SAP is a plant GP-like protein which participate in the enzymatic system aimed at scavenging oxygen free-radicals in plants.

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases.

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.

Serine incorporation into the selenocysteine moiety of glutathione peroxidase.

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.