Anti-apoptotic activity of the glutathione peroxidase homologue encoded by HIV-1

The third reading frame of the envelope gene from HIV-1 codes for a protein homologous to the human selenoprotein glutathione peroxidase (GPX). Cells stably or transiently transfected with a HIV-1 GPX construct are protected against the loss of the mitochondrial transmembrane potential and subsequent cell death induced by exogenous reactive oxygen species (ROS) as well as mitochondrion-generated ROS. However, HIV-1 GPX does not confer a general apoptosis resistance, because HIV-1 GPX-transfected cells were not protected against cell death induced by staurosporine or oligomycin. The inhibition of cell death induced by the ROS donor tert-butylhydroperoxide was also observed in cells depleted from endogenous glutathione (GSH), suggesting that GSH is not the sole electron acceptor for HIV-1 GPX. Clinical HIV-1 isolates from long-term non-progressors (untreated patients with diagnosed HIV-1 infection for > 10 years, with CD4 T cell count of > 500 cells/mm3) mostly possess an intact GPX gene (with only 18% of loss-of-function mutations), while HIV-1 isolates from patients developing AIDS contain non-functional GPX mutants in 9 out of 17 cases (53%). Altogether, these data suggest that HIV-1 GPX possesses a cytoprotective, pathophysiologically relevant function.     

Selenium-dependent glutathione peroxidase modules encoded by RNA viruses

Glutathione peroxidase (GPx) is the prototypical eukaryotic selenoprotein, with the rare amino acid selenocysteine (Sec) at the enzyme active site, encoded by the UGA codon in RNA. A DNA virus,Molluscum contagiosum, has now been shown to encode a functional selenium-dependent GPx enzyme. Using modifications of conventional sequence database searching techniques to locate potential viral GPx modules, combined with structurally guided comparative sequence analysis, we provide compelling evidence that Se-dependent GPx modules are encoded in a number of RNA viruses, including potentially serious human pathogens like HIV-1 and hepatitis C virus, coxsackievirus B3, HIV-2, and measles virus. Analysis of the sequences of multiple viral isolates reveals conservation of the putative GPx-related features, at least within viral subtypes or genotypes, supporting the hypothesis that these are functional GPx modules.     

Kinetic studies on the glutathione peroxidase activity of selenium-containing glutathione transferase

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.     

Further electrophoretic studies of erythrocyte glutathione peroxidase.

A new, inexpensive technique has been devised for the detection of glutathione peroxidase after electrophoresis. Surveys of several racial groups have revealed new variant phenotypes at polymorphic frequencies in African and Lebanese populations.     

Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein

For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx.     

Functional role of the HIV-1 Rev exon 1 encoded region in complex formation and trans-dominant inhibition

To study functional aspects of the exon 1 encoded region of the human immunodeficiency virus type 1 Rev protein, the viral Tev protein which exhibits low Rev activity but lacks the rev exon 1 encoded region was examined. Neither Rev-Tev heteromer complex formation nor inhibition of Rev by an export deficient Tev mutant was observed. Insertion of the rev exon 1 encoded region into the Tev mutant allowed it to oligomerize with Rev and act as a trans-dominant negative mutant. This showed that the exon 1 encoded region of Rev is essential for oligomerization and that oligomerization is a prerequisite for trans-dominant inhibition.     

Blood glutathione peroxidase

Biological Trace Element Research - Manual and automated assays for the determination of glutathione peroxidase activity in bovine, sheep, pig, and human blood samples are described. The...     

Gastrointestinal glutathione peroxidase

The gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the GPx family. In rodents, it is exclusively expressed in the gastrointestinal tract, in humans also in liver. It has, therefore, been discussed to function as a primary barrier against the absorption of ingested hydroperoxides. A vital function of GI-GPx can be deduced from the unusual stability of its mRNA under selenium-limiting conditions, the presence of low amounts of GI-GPx protein in selenium deficiency where cGPx is absent, and the fast reappearance of the GI-GPx protein upon refeeding of cultured cells with selenium compared to the slower reappearance of cGPx protein. Furthermore, the Secis efficiency of GI-GPx is low when compared to cGPx and PHGPx. It is, however, almost independent of the selenium status of the cells tested. All these characteristics rank GI-GPx high in the hierarchy of selenoproteins and point to a role of GI-GPx which might be more crucial than that of cGPx, at least in the gastrointestinal system.     

The Drosophila glutathione S-transferase 1-1 is encoded by an intronless gene at 87B

The Drosophila glutathione S-transferase 1-1 is a dimer of a 209 amino acid subunit, designated DmGST1. DmGST1 is encoded by a member of a multigene family. Sequence analysis of a genomic clone for GST1 revealed that it is encoded by an intronless gene. We designate this gene and its other family members the GST D genes in the glutathione S-transferase gene superfamily. The Drosophila GST D genes are mapped by in situ hybridization to chromosome 3R at 87B of the polytene chromosome, which is flanked by the two clusters of hsp70 genes at 87A7 and 87C1. Cytogenetic data in the literature indicated that a puff occurred in this region under heat shock. We report that the glutathione S-transferase activity in Kco cells as determined by conjugation with 1-chloro-2,4-dinitrobenzene is elevated slightly to two-fold under heat shock. The implication of this finding is discussed.     

Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats

Severe steroidogenic and spermatogenic alterations are reported in association with diabetic manifestations in humans and experimental animals. This study was planned to determine whether oxidative stress is involved in diabetes-induced alterations in the testes. Diabetes was induced in male rats by injection of 50 mg/kg of streptozotocin (STZ). Ten weeks after injection of STZ, levels of selenium and activities of selenium dependent-glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were measured in rat testis. Lipid and protein oxidations were evaluated as measurements of testis malondialdehyde (MDA) and protein carbonyl levels, respectively. Testis sulfydryl (SH) levels were also determined. The control levels of GPx and PHGPx activities were found to be 46.5 +/- 6.2 and 108.8 +/- 19.8 nmol GSH/mg protein/min, respectively. Diabetes caused an increase in testis GPx (65.0 +/- 21.1) and PHGPx (155.9 +/- 43.1) activities but did not affect the levels of selenium or SH. However, the testis MDA and protein carbonyl levels as markers of lipid and protein oxidation, respectively, did not increase in the diabetic group. Aminoguanidine (AG) treatment of diabetic rats returned the testis PHGPx activity (136.5 +/- 24.9) to the control level but did not change the value of GPx activity (69.2 +/- 17.4) compared with diabetic group. MDA and protein carbonyl levels in testis were not affected by AG treatment of diabetic rats, but interestingly AG caused SH levels to increase. The results indicate that reactive oxygen radicals were not involved in possible testicular complications of diabetes because diabetes-induced activations of GPx and PHGPx provided protection against oxidative stress, which was reported to be related to some diabetic complications.     

Involvement of mitochondrial phospholipid hydroperoxide glutathione peroxidase as an antiapoptotic factor

Although reactive oxygen species (ROS) such as superoxide and hydroperoxide are known to induce apoptotic cell death, little is known as to the apoptotic death signaling of mitochondrial ROS. Recent evidence has suggested that antioxidant enzymes in mitochondria may be responsible for the regulation of cytochrome c release and apoptotic cell death. This paper examines the current state of knowledge regarding the role of mitochondrial antioxidant enzymes, especially phospholipid hydroperoxide glutathione peroxidase. A model for the release of cytochrome c by lipid hydroperoxide has also been proposed.     

Molecular simulation studies of a selenium-containing scFv catalytic antibody that mimics glutathione peroxidase

GPX is a mammalian antioxidant selenoenzyme which protects biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides (ROOH), using Glutathione (GSH) as the reducing substrate. The single-chain Fv fragment of the monoclonal antibody 2F3 (scFv2F3) can be converted into the selenium-containing Se-scFv2F3 by chemical modification of the serine. The new selenium-containing catalytic antibody Se-scFv2F3 acts as a glutathione peroxidase (GPX) mimic with high catalytic efficiency.In order to investigate which residue of scFv2F3 is converted into selenocysteine and to describe the proper reaction site of GSH to Se-scFv2F3, a three-dimensional structure of scFv2F3 is built by means of homology modeling. The 3D model is assessed by molecular dynamics (MD) simulation to determine its stability and by comparison with those of known protein structures. After the serine in the scFv2F3 is modified to selenocysteine, a catalytic antibody (abzyme) is obtained. From geometrical considerations, the solvent-accessible surface of the protein is examined. The computer-aided docking and energy minimization (EM) calculations of the abzyme–GSH complex are then carried out to explore the possible active site of the glutathione peroxidase mimic Se-scFv2F3. The structural information from the theoretically modeled complex can help us to further understand the catalytic mechanism of GPX.     

Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals

Background  

Phospholipid hydroperoxide glutathione peroxidases (PHGPx), the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown.     

Characteristics of human milk glutathione peroxidase

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.     

Phylogenetic distribution of glutathione peroxidase.

1. The enzyme glutathione peroxidase (E.C.1.11.1.9), known to be a selenoprotein from mammalian sources, was detected in the following vertebrates: fish, frog, salamander, and turtle. 2. Among invertebrates, the enzyme was detected in crayfish and snail but not in insects or earthworm. 3. No plant tissues or microorganisms showed any evidence of the enzyme activity. 4. The presence of the enzyme activity in so many animal groups implies the widespread occurrence of genetic information for the specific assimilation of the selenium atom.     

Isolation and purification of glutathione peroxidase

Electrophoretically homogeneous glutathione peroxidase (EC 1.11.1.9) preparation from rat liver with a specific activity of 1.46 U/mg of protein and a yield of 7.2% was obtained using the purification procedure developed. The K(M) values for reduced glutathione and hydrogen peroxide were 0.033 and 0.208 mM, respectively. The enzymatic reaction had the following characteristics: the temperature optimum, 32 degrees C; the pH optimum, 7.4; and the activation energy, 29.1 kJ/mol. The molecular weight of the enzyme was 88 kDa.