Omega-class Glutathione Transferases Research Articles

Rattus norvegicus Glutathione Transferase Omega 1 Localization in Oral Tissues and Interactions with Food Phytochemicals.

Glutathione transferases are xenobiotic-metabolizing enzymes with both glutathione-conjugation and ligandin roles. GSTs are present in chemosensory tissues and fluids of the nasal/oral cavities where they protect tissues from exogenous compounds, including food molecules. In the present study, we explored the presence of the omega-class glutathione transferase (GSTO1) in the rat oral cavity. Using immunohistochemistry, GSTO1 expression was found in taste bud cells of the tongue epithelium and buccal cells of the oral epithelium. Buccal and lingual extracts exhibited thiol-transferase activity (4.9 ± 0.1 and 1.8 ± 0.1 μM/s/mg, respectively). A slight reduction from 4.9 ± 0.1 to 4.2 ± 0.1 μM/s/mg (p < 0.05; Student's t test) was observed in the buccal extract with 100 μM GSTO1-IN-1, a specific inhibitor of GSTO1. RnGSTO1 exhibited the usual activities of omega GSTs, i.e., thiol-transferase (catalytic efficiency of 8.9 × 104 M-1·s-1), and phenacyl-glutathione reductase (catalytic efficiency of 8.9 × 105 M-1·s-1) activities, similar to human GSTO1. RnGSTO1 interacts with food phytochemicals, including bitter compounds such as luteolin (Ki = 3.3 ± 1.9 μM). Crystal structure analysis suggests that luteolin most probably binds to RnGSTO1 ligandin site. Our results suggest that GSTO1 could interact with food phytochemicals in the oral cavity.

Identification of Omega-class glutathione transferases in helminths of the Taeniidae family

Glutathione transferase enzymes (GSTs) are believed to be a major detoxification system in helminth parasites and have been associated with immunomodulation of the host response. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least five different GSTs, but no Omega-class enzymes have been reported in this parasite or in any other cestode. Herein we report the identification of a new member of the GST superfamily in E. granulosus s.l., which is phylogenetically related to the Omega-class: EgrGSTO. Through mass spectrometry, we showed that the 237 amino acids protein EgrGSTO is expressed by the parasite. Moreover, we identified homologues of EgrGSTO in other eight members of the Taeniidae family, including E. canadensis, E. multilocularis, E. oligarthrus, Hydatigera taeniaeformis, Taenia asiatica, T. multiceps, T. saginata and T. solium. A manual sequence inspection and rational modification yielded eight Taeniidae’s GSTO sequences, each one encoding for a 237 aa polypeptide showing 80.2% overall identity. To the best of our knowledge, this is the first description of genes encoding for Omega-class GSTs in worms belonging to the Taeniidae family -that at least in E. granulosus s.l. is expressed as a protein- suggesting the gene encodes for a functional protein.

Omega-Class Glutathione Transferases Protect DNA from Oxidative Stress in Pathogenic Helminth Reproductive Cells

Pathogenic helminths have evolved mechanisms to preserve reproductive function while surviving long-term in the host via robust protective responses. A protective role of antioxidant enzymes in preventing DNA degradation has long been proposed, but little evidence has been provided. Here, we show that omega-class glutathione transferases (GSTOs) are critical for maintaining viability by protecting the reproductive cell DNA of the carcinogenic liver fluke, Clonorchis sinensis. Clonorchis sinensis GSTO (CsGSTO) activities modified by changes in the GSH/GSSG and NADPH/NADP+ molar ratios suppressed the overproduction of reactive oxygen species. CsGSTO1 and CsGSTO2 catalyzed deglutathionylation under physiologic and low-stress conditions (GSH/GSSG ratio of 6:1 or higher) but promoted glutathionylation under high-stress conditions (GSH/GSSG ratio of 3:1 or lower). Gliotoxin-induced functional disruption of CsGSTOs in living C. sinensis reduced the GSH/GSSG molar ratio and increased the production of protein glutathionylation (PSSG) under physiologic and low-stress conditions, indicating that suppression of GSTO function did not affect deglutathionylation. However, the perturbation of CsGSTOs decreased the GSH/GSSG ratio but also reduced PSSG production under high oxidative stress, demonstrating that glutathionylation was impeded. In response to oxidative stimuli, C. sinensis decreased GSTO-specific dehydroascorbate reductase and thiol transferase activities and the GSH/GSSG ratio, while it increased the NADPH/NADP+ ratio and PSSG. CsGSTOs utilized GSH to regulate GSH/GSSG and NADPH/NADP+ recycling and triggered a redox signal leading to nuclear translocation. Nuclear-imported CsGSTOs were modified by glutathionylation to prevent DNA damage. Antibodies specific to CsGSTOs dose-dependently inhibited this process. Disruption of CsGSTOs or the depletion of GSH caused glutathionylation defects, leading to DNA degradation. Our results demonstrate that CsGSTOs and the GSH system play a previously unappreciated role in protecting DNA from oxidative stress.

Proposed mechanism for monomethylarsonate reductase activity of human omega-class glutathione transferase GSTO1-1

Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

Omega-Class Glutathione Transferases of Carcinogenic Liver Fluke, Clonorchis sinensis, Modulate Apoptosis and Differentiation of Host Cholangiocytes.

The small liver fluke Clonorchis sinensis causes hepatobiliary ductal infections in humans. Clonorchiasis is characterized histopathologically by ductal dysplasia, hyperplasia and metaplasia, which closely resembles cholangiocarcinoma (CCA). The disruption of programmed cell death is critical for malignant transformation, while molecular events underlying these phenomena have poorly been understood in clonorchiasis-related CCA tumorigenesis. We incorporated recombinant C. sinensis omega-class glutathione transferase (rCsGSTo) 1 or 2 into human intrahepatic biliary epithelial cells (HIBECs) and analyzed pathophysiological alterations of HIBECs upon the application of oxidative stress. rCsGSTos partially but significantly rescued HIBECs from cell death by inhibiting oxidative stress-induced apoptosis (p < 0.01). rCsGSTos modulated transcriptional levels of numerous genes. We analyzed 13 genes involved in programmed cell death (the upregulation of five antiapoptotic and two apoptotic genes, and the downregulation of one antiapoptotic and five apoptotic genes) and 11 genes associated with cell differentiation (the increase in seven and decrease in four genes) that showed significant modifications (p < 0.05). The induction profiles of the mRNA and proteins of these differentially regulated genes correlated well with each other, and mostly favored apoptotic suppression and/or cell differentiation. We detected increased active, phosphorylated forms of Src, PI3K/Akt, NF-κB p65, MKK3/6 and p38 MAPK, but not JNK and ERK1/2. CsGSTos were localized in the C. sinensis-infected rat cholangiocytes, where cytokeratin 19 was distributed. Our results demonstrated that CsGSTos excreted to the biliary lumen are internalized and accumulated in the host cholangiocytes. When cholangiocytes underwent oxidative stressful condition, CsGSTos appeared to be critically involved in both antiapoptotic process and the differentiation of host cholangiocytes through the regulation of target genes following the activation of responsible signal molecules.

Glutathione transferase Omega 1 confers protection against azoxymethane-induced colorectal tumour formation.

Inflammatory bowel disease (IBD) is characterized by multiple alterations in cytokine expression and is a risk factor for colon cancer. The Omega class glutathione transferase GSTO1-1 regulates the release of the pro-inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) by deglutathionylating NEK7 in the NLRP3 inflammasome. When treated with azoxymethane and dextran sodium sulphate (AOM/DSS) as a model of IBD, Gsto1-/- mice were highly sensitive to colitis and showed a significant increase in the size and number of colon tumours compared with wild-type (WT) mice. Gsto1-/- mice treated with AOM/DSS had significantly lower serum IL-1β and IL-18 levels as well as significantly decreased interferon (IFN)-γ, decreased pSTAT1 and increased pSTAT3 levels in the distal colon compared with similarly treated WT mice. Histologically, AOM/DSS treated Gsto1-/- mice showed increased active chronic inflammation with macrophage infiltration, epithelial dysplasia and invasive adenocarcinoma compared with AOM/DSS treated WT mice. Thus, this study shows that GSTO1-1 regulates IL-1β and IL-18 activation and protects against colorectal cancer formation in the AOM/DSS model of IBD. The data suggest that while GSTO1-1 is a new target for the regulation of the NLRP3 inflammasome-associated cytokines IL-1β and IL-18 by small molecule inhibitors, there is a possibility that anti-inflammatory drugs targeting these cytokines may potentiate colon cancer in some situations.

GSTO1*CC Genotype (rs4925) Predicts Shorter Survival in Clear Cell Renal Cell Carcinoma Male Patients.

Omega class glutathione transferases, GSTO1-1 and GSTO2-2, exhibit different activities involved in regulation of inflammation, apoptosis and redox homeostasis. We investigated the the prognostic significance of GSTO1 (rs4925) and GSTO2 (rs156697 and rs2297235) polymorphisms in clear cell renal cell carcinoma (ccRCC) patients. GSTO1-1 and GSTO2-2 expression and phosphorylation status of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ /mammalian target of rapamycin (mTOR) and Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathways in non-tumor and tumor ccRCC tissue, as well as possible association of GSTO1-1 with signaling molecules were also assessed. GSTO genotyping was performed by quantitative PCR in 228 ccRCC patients, while expression and immunoprecipitation were analyzed by Western blot in 30 tissue specimens. Shorter survival in male carriers of GSTO1*C/C wild-type genotype compared to the carriers of at least one variant allele was demonstrated (p = 0.049). GSTO1*C/C genotype independently predicted higher risk of overall mortality among male ccRCC patients (p = 0.037). Increased expression of GSTO1-1 and GSTO2-2 was demonstrated in tumor compared to corresponding non-tumor tissue (p = 0.002, p = 0.007, respectively), while GSTO1 expression was correlated with interleukin-1β (IL-1β)/pro-interleukin-1β (pro-IL-1β) ratio (r = 0.260, p = 0.350). Interaction of GSTO1 with downstream effectors of investigated pathways was shown in ccRCC tumor tissue. This study demonstrated significant prognostic role of GSTO1 polymorphism in ccRCC. Up-regulated GSTO1-1 and GSTO2-2 in tumor tissue might contribute to aberrant ccRCC redox homeostasis.

Clonorchis sinensis omega-class glutathione transferases are reliable biomarkers for serodiagnosis of clonorchiasis and opisthorchiasis

ObjectivesTo determine the potential for immunodiagnostic application of two recombinant forms of Clonorchis sinensis omega-class glutathione transferases (rCsGSTo1 and rCsGSTo2) against human small liver-fluke C. sinensis and Opisthorchis viverrini infections. MethodsSpecific antibody levels against rCsGSTo1 and rCsGSTo2 in patients' sera of egg-positive opisthorchiasis (n = 87) and clonorchiasis (n = 120), as well as those in sera from patients with other helminthic infections (n = 252) and healthy controls (n = 40) were retrospectively analysed by ELISA. ResultsWe observed highly positive correlation coefficients between specific antibody levels against rCsGSTo1 and rCsGSTo2 and egg counts per gramme of faeces (EPG) of patients with opisthorchiasis (n = 87; r = 0.88 for rCsGSTo1 and r = 0.90 for rCsGSTo2). Sera from opisthorchiasis patients whose EPG counts >100 (n = 43) revealed high antibody titres against both antigens. Patients' sera with low EPG counts (<100, n = 44) also exhibited reliable sensitivities of 93.2% and 97.7% for rCsGSTo1 and rCsGSTo2, respectively. Sera from clonorchiasis patients showed sensitivities of 90% (108/120 samples) and 89.2% (107/120 sera) for rCsGSTo1 and rCsGSTo2. Overall diagnostic sensitivities for liver-fluke infections were 92.3% for rCsGSTo1 (191/207 samples) and 93.2% for rCsGSTo2 (193/207 samples). Specificities were 89.7% (rCsGSTo1) and 97.6% (rCsGSTo2). ConclusionsDetection of specific antibody levels against rCsGSTo1 or rCsGSTo2 might be promising for the serodiagnosis of patients infected with these two phylogenetically close carcinogenic liver-flukes.

Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress.

BackgroundClonorchis sinensis causes a major food-borne helminthic infection. This species locates in mammalian hepatobiliary ducts, where oxidative stressors and hydrophobic substances are profuse. To adapt to the hostile micromilieu and to ensure its long-term survival, the parasite continuously produces a diverse repertoire of antioxidant enzymes including several species of glutathione transferases (GSTs). Helminth GSTs play pertinent roles during sequestration of harmful xenobiotics since most helminths lack the cytochrome P-450 detoxifying enzyme.MethodsWe isolated and analyzed the biochemical properties of two omega-class GSTs of C. sinensis (CsGSTo1 and CsGSTo2). We observed spatiotemporal expression patterns in accordance with the maturation of the worm’s reproductive system. Possible biological protective roles of CsGSTos in these organs under oxidative stress were investigated.ResultsThe full-length cDNAs of CsGSTo1 and 2 constituted 965 bp and 1,061 bp with open reading frames of 737 bp (246 amino acids) and 669 bp (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal α-helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44 % sequence identity with each other and 14.8–44.8 % with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. S-hexylglutathione potently and competitively inhibited the active-site at nanomolar concentrations (0.63 and 0.58 nM for rCsGSTo1 and 2). Interestingly, rCsGSTos exhibited high enzyme activity toward mu- and theta-class GST specific substrate, 4-nitrobenzyl chloride. Expression of CsGSTo transcripts and proteins increased beginning in 2-week-old juveniles and reached their highest levels in 4-week-old adults. The proteins were mainly expressed in the elements of the reproductive system, such as vitelline follicles, testes, seminal receptacle, sperm and eggs. Oxidative stressors induced upregulated expression of CsGSTos in these organs. Regardless of oxidative stresses, CsGSTos continued to be highly expressed in eggs. CsGSTo1 or 2 overexpressing bacteria demonstrated high resistance under oxidative killing.ConclusionsCsGSTos might be critically involved in protection of the reproductive system during maturation of C. sinensis worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1622-2) contains supplementary material, which is available to authorized users.

Structure, function and disease relevance of Omega-class glutathione transferases.

The Omega-class cytosolic glutathione transferases (GSTs) have distinct structural and functional attributes that allow them to perform novel roles unrelated to the functions of other GSTs. Mammalian GSTO1-1 has been found to play a previously unappreciated role in the glutathionylation cycle that is emerging as significant mechanism regulating protein function. GSTO1-1-catalyzed glutathionylation or deglutathionylation of a key signaling protein may explain the requirement for catalytically active GSTO1-1 in LPS-stimulated pro-inflammatory signaling through the TLR4 receptor. The observation that ML175 a specific GSTO1-1 inhibitor can block LPS-stimulated inflammatory signaling has opened a new avenue for the development of novel anti-inflammatory drugs that could be useful in the treatment of toxic shock and other inflammatory disorders. The role of GSTO2-2 remains unclear. As a dehydroascorbate reductase, it could contribute to the maintenance of cellular redox balance and it is interesting to note that the GSTO2 N142D polymorphism has been associated with multiple diseases including Alzheimer's disease, Parkinson's disease, familial amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, age-related cataract and breast cancer.

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

Three-dimensional structure of a Bombyx mori Omega-class glutathione transferase

Glutathione transferases (GSTs) are major phase II detoxification enzymes that play central roles in the defense against various environmental toxicants as well as oxidative stress. Here we report the crystal structure of an Omega-class glutathione transferase of Bombyx mori, bmGSTO, to gain insight into its catalytic mechanism. The structure of bmGSTO complexed with glutathione determined at a resolution of 2.5Å reveals that it exists as a dimer and is structurally similar to Omega-class GSTs with respect to its secondary and tertiary structures. Analysis of a complex between bmGSTO and glutathione showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTO mutants indicated that amino acid residues Leu62, Lys65, Lys77, Val78, Glu91 and Ser92 in the G-site contribute to catalytic activity.

Structural Insights into Omega-Class Glutathione Transferases: A Snapshot of Enzyme Reduction and Identification of a Non-Catalytic Ligandin Site

Glutathione transferases (GSTs) are dimeric enzymes containing one active-site per monomer. The omega-class GSTs (hGSTO1-1 and hGSTO2-2 in humans) are homodimeric and carry out a range of reactions including the glutathione-dependant reduction of a range of compounds and the reduction of S-(phenacyl)glutathiones to acetophenones. Both types of reaction result in the formation of a mixed-disulfide of the enzyme with glutathione through the catalytic cysteine (C32). Recycling of the enzyme utilizes a second glutathione molecule and results in oxidized glutathione (GSSG) release. The crystal structure of an active-site mutant (C32A) of the hGSTO1-1 isozyme in complex with GSSG provides a snapshot of the enzyme in the process of regeneration. GSSG occupies both the G (GSH-binding) and H (hydrophobic-binding) sites and causes re-arrangement of some H-site residues. In the same structure we demonstrate the existence of a novel “ligandin” binding site deep within in the dimer interface of this enzyme, containing S-(4-nitrophenacyl)glutathione, an isozyme-specific substrate for hGSTO1-1. The ligandin site, conserved in Omega class GSTs from a range of species, is hydrophobic in nature and may represent the binding location for tocopherol esters that are uncompetitive hGSTO1-1 inhibitors.

Structural Insights into the Dehydroascorbate Reductase Activity of Human Omega-Class Glutathione Transferases

The reduction of dehydroascorbate (DHA) to ascorbic acid (AA) is a vital cellular function. The omega-class glutathione transferases (GSTs) catalyze several reductive reactions in cellular biochemistry, including DHA reduction. In humans, two isozymes (GSTO1-1 and GSTO2-2) with significant DHA reductase (DHAR) activity are found, sharing 64% sequence identity. While the activity of GSTO2-2 is higher, it is significantly more unstable in vitro. We report the first crystal structures of human GSTO2-2, stabilized through site-directed mutagenesis and determined at 1.9 Å resolution in the presence and absence of glutathione (GSH). The structure of a human GSTO1-1 has been determined at 1.7 Å resolution in complex with the reaction product AA, which unexpectedly binds in the G-site, where the glutamyl moiety of GSH binds. The structure suggests a similar mode of ascorbate binding in GSTO2-2. This is the first time that a non-GSH-based reaction product has been observed in the G-site of any GST. AA stacks against a conserved aromatic residue, F34 (equivalent to Y34 in GSTO2-2). Mutation of Y34 to alanine in GSTO2-2 eliminates DHAR activity. From these structures and other biochemical data, we propose a mechanism of substrate binding and catalysis of DHAR activity.

The secretory omega‐class glutathione transferase OvGST3 from the human pathogenic parasite Onchocerca volvulus

Onchocerciasis or river blindness, caused by the filarial nematode Onchocerca volvulus, is the second leading cause of blindness due to infectious diseases. The protective role of the omega-class glutathione transferase 3 from O. volvulus (OvGST3) against intracellular and environmental reactive oxygen species has been described previously. In the present study, we continue our investigation of the highly stress-responsive OvGST3. Alternative splicing of two exons and one intron retention generates five different transcript isoforms that possess a spliced leader at their 5'-end, indicating that the mechanism of mature mRNA production involves alternative-, cis- and trans-splicing processes. Interestingly, the first two exons of the ovgst3 gene encode a signal peptide before sequence identity to other omega-class glutathione transferases begins. Only the recombinant expression of the isoform that encodes the longest deduced amino acid sequence (OvGST3/5) was successful, with the purified enzyme displaying modest thiol oxidoreductase activity. Significant IgG1 and IgG4 responses against recombinantly expressed OvGST3/5 were detected in sera from patients with the generalized as well as the chronic hyperreactive form of onchocerciasis, indicating exposure of the secreted protein to the human host's immune system and its immunogenicity. Immunohistological localization studies performed at light and electron microscopy levels support the extracellular localization of the protein. Intensive labeling of the OvGST3 was observed in the egg shell at the morula stage of the embryo, indicating extremely defined, stage-specific expression for a short transient period only.

Deletion of Glu155 causes a deficiency of glutathione transferase Omega 1-1 but does not alter sensitivity to arsenic trioxide and other cytotoxic drugs

The Omega class glutathione transferase GSTO1-1 can catalyze the reduction of pentavalent methylated arsenic species and is responsible for the biotransfomation of potentially toxic α-haloketones. We investigated the cause of GSTO1-1 deficiency in the T-47D breast cancer cell line and found that the cell line is hemizygous for a polymorphic allele that encodes the deletion of Glu155. Northern and Western blots show that T-47D cells contain GSTO1 mRNA but no GSTO1-1 protein suggesting that the deletion of Glu155 causes GSTO1-1 deficiency in vivo. In further support of this contention we found that lymphoblastoid cell lines from subjects who are heterozygous for the deletion of Glu155 have only 60% of normal activity with the GSTO1-1 specific substrate 4-nitrophenacyl glutathione. Pulse-chase studies showed that the deletion of Glu155 causes increased turnover of GSTO1-1 in T47-D cells. These data establish the fact that the polymorphic deletion of Glu155 can cause GSTO1-1 deficiency in vivo. GSTO1-1 expression is elevated in some cell lines that are resistant to the cytotoxic cancer drugs adriamycin, etoposide and cisplatinum but its specific contribution to multi drug resistance has not been evaluated. In this study GSTO1-1 deficient T47-D cells were used to determine if GSTO1-1 contributes directly to arsenic and drug resistance. We established stable expression of normal GSTO1-1 in T-47D cells and found that this did not alter sensitivity to arsenic trioxide, cisplatinum daunorubicin or etoposide.

Oxidative stress inCaenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO‐1)

To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO-1 from Caenorhabditis elegans (GSTO-1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO-1 provided a test of resistance to long-term exposure under oxidative stress. After affinity purification, the recombinant GSTO-1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO-1-promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an approximately 300-nucleotide sequence upstream of the ATG start site necessary for GSTO-1 expression. Site-specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt-2 indicated the involvement of this gut-specific transcription factor in GSTO-1 expression. Transcriptional up-regulation under stress conditions of GSTO-1 was confirmed by analyzing promoter-reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO-1 in vivo, transgenic animals overexpressing GSTO-1 were generated exhibiting an increased resistance to juglone-, paraquat-, and cumene hydroperoxide-induced oxidative stress. Specific silencing of the GSTO-1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress-responsive GSTO-1 plays a key role in counteracting environmental stress.

Glutathione Transferase Omega 1 Catalyzes the Reduction of S-(Phenacyl)glutathiones to Acetophenones

S-(Phenacyl)glutathione reductase (SPG-R) plays a significant role in the biotransformation of reactive alpha-haloketones to nontoxic acetophenones. Comparison of the apparent subunit size, amino acid composition, and catalysis of the reduction of S-(phenacyl)glutathiones indicated that a previously described rat SPG-R (Kitada, M., McLenithan, J. C., and Anders, M. W. (1985) J. Biol. Chem. 260, 11749-11754) is homologous to the omega-class glutathione transferase GSTO1-1. The available data show that the SPG-R reaction is catalyzed by GSTO1-1 and not by other GSTs, including the closely related GSTO2-2 isoenzyme. In the proposed reaction mechanism, the active-site cysteine residue of GSTO1-1 reacts with the S-(phenacyl)glutathione substrate to give an acetophenone and a mixed disulfide with the active-site cysteine; a second thiol substrate (e.g., glutathione or 2-mercaptoethanol) reacts with the active-site disulfide to regenerate the catalytically active enzyme and to form a mixed disulfide. A new spectrophotometric assay was developed that allows the rapid determination of SPG-R activity and specific measurement of GSTO1-1 in the presence of other GSTs. This is the first specific reaction attributed to GSTO1-1, and these results demonstrate the catalytic diversity of GSTO1-1, which, in addition to SPG-R activity, catalyzes the reduction of dehydroascorbate and monomethylarsonate(V) and also possesses thioltransferase and GST activity.

A Peroxisomal Glutathione Transferase of Saccharomyces cerevisiae Is Functionally Related to Sulfur Amino Acid Metabolism

Saccharomyces cerevisiae cells contain three omega-class glutathione transferases with glutaredoxin activity (Gto1, Gto2, and Gto3), in addition to two glutathione transferases (Gtt1 and Gtt2) not classifiable into standard classes. Gto1 is located at the peroxisomes, where it is targeted through a PTS1-type sequence, whereas Gto2 and Gto3 are in the cytosol. Among the GTO genes, GTO2 shows the strongest induction of expression by agents such as diamide, 1-chloro-2,4-dinitrobenzene, tert-butyl hydroperoxide or cadmium, in a manner that is dependent on transcriptional factors Yap1 and/or Msn2/4. Diamide and 1-chloro-2,4-dinitrobenzene (causing depletion of reduced glutathione) also induce expression of GTO1 over basal levels. Phenotypic analyses with single and multiple mutants in the S. cerevisiae glutathione transferase genes show that, in the absence of Gto1 and the two Gtt proteins, cells display increased sensitivity to cadmium. A gto1-null mutant also shows growth defects on oleic acid-based medium, which is indicative of abnormal peroxisomal functions, and altered expression of genes related to sulfur amino acid metabolism. As a consequence, growth of the gto1 mutant is delayed in growth medium without lysine, serine, or threonine, and the mutant cells have low levels of reduced glutathione. The role of Gto1 at the S. cerevisiae peroxisomes could be related to the redox regulation of the Str3 cystathionine beta-lyase protein. This protein is also located at the peroxisomes in S. cerevisiae, where it is involved in transulfuration of cysteine into homocysteine, and requires a conserved cysteine residue for its biological activity.

Glutaredoxins in fungi.

Glutaredoxins (GRXs) can be subdivided into two subfamilies: dithiol GRXs with the CPY/FC active site motif, and monothiol GRXs with the CGFS motif. Both subfamilies share a thioredoxin-fold structure. Some monothiol GRXs exist with a single-Grx domain while others have a thioredoxin-like domain (Trx) and one or more Grx domains in tandem. Most fungi have both dithiol and monothiol GRXs with different subcellular locations. GRX-like molecules also exist in fungi that differ by one residue from one of the canonical active site motifs. Additionally, Omega-class glutathione transferases (GSTs) are active as GRXs. Among fungi, the GRXs more extensively studied are those from Saccharomyces cerevisiae. This organism contains two dithiol GRXs (ScGrx1 and ScGrx2) with partially overlapping functions in defence against oxidative stress. In this function, they cooperate with GSTs Gtt1 and Gtt2. While ScGrx1 is cytosolic, two pools exist for ScGrx2, a major one at the cytosol and a minor one at mitochondria. On the other hand, S. cerevisiae cells have two monothiol GRXs with the Trx-Grx structure (ScGrx3 and ScGrx4) that locate at the nucleus and probably regulate the activity of transcription factors such as Aft1, and one monothiol GRX with the Grx structure (ScGrx5) that localizes at the mitochondrial matrix, where it participates in the synthesis of iron-sulphur clusters. The function of yeast Grx5 seems to be conserved along the evolutionary scale.