GSH Transferase Research Articles

Physiology, Biochemistry, and Transcriptomics Jointly Reveal the Phytotoxicity Mechanism of Acetochlor on Pisum sativum L.

Acetochlor, as a commonly used pre-emergent herbicide, can be toxic to crops and affect production if used improperly. However, the toxic mechanism of acetochlor on plants is not fully understood. The present study used a combination of transcriptomic analysis and physiological measurements to investigate the effects of short-term (15-day) exposure to different concentrations of acetochlor (1, 10, 20 mg/kg) on the morphology, physiology, and transcriptional levels of pea seedlings, aiming to elucidate the toxic response and resistance mechanisms in pea seedlings under herbicide stress. The results showed that the toxicity of acetochlor to pea seedlings was dose-dependent, manifested as dwarfing and stem base browning with increasing concentrations, especially at 10 mg/kg and above. Analysis of the antioxidant system showed that from the 1 mg/kg treatment, malondialdehyde, superoxide dismutase, peroxidase, and glutathione peroxidase in peas increased with increasing concentrations of acetochlor, indicating oxidative damage. Analysis of the glutathione (GSH) metabolism system showed that under 10 mg/kg treatment, the GSH content of pea plants significantly increased, and GSH transferase activity and gene expression were significantly induced, indicating a detoxification response in plants. Transcriptomic analysis showed that after acetochlor treatment, differentially expressed genes in peas were significantly enriched in the phenylpropane metabolic pathway, and the levels of key metabolites (flavonoids and lignin) were increased. In addition, we found that acetochlor-induced dwarfing of pea seedlings may be related to gibberellin signal transduction. Environ Toxicol Chem 2024;43:2005-2019. © 2024 SETAC.

Dietary and Lifestyle Factors Modulate the Activity of the Endogenous Antioxidant System in Patients with Age-Related Macular Degeneration: Correlations with Disease Severity.

Age-related macular degeneration (AMD) is a common cause of blindness in the elderly population, but the pathogenesis of this disease remains largely unknown. Since oxidative stress is suggested to play a major role in AMD, we aimed to assess the activity levels of components of the antioxidant system in patients with AMD. We also investigated whether lifestyle and dietary factors modulate the activity of these endogenous antioxidants and clinical parameters of disease severity. We recruited 330 patients with AMD (39 with early, 100 with intermediate and 191 with late form of AMD) and 121 controls in this study. At enrolment, patients’ dietary habits and physical activity were assessed, and each study participant underwent a thorough ophthalmologic examination. The activity of several components of the antioxidant system were measured in red blood cells and platelets using both kinetic and spectrophotometric methods. Patients with AMD consumed much lower levels of fatty fish and eggs than the control group (p = 0.008 and p = 0.04, respectively). In the nAMD group, visual acuity (VA) correlated positively with green vegetable consumption (Rs = +0.24, p = 0.004) and omega-3-rich oil intake (Rs = +0.17, p = 0.03). In the AMD group, the total physical activity MET score correlated positively with VA (Rs = +0.17, p = 0.003) and correlated negatively with the severity of AMD (Rs = −0.14, p = 0.01). A multivariate analysis of patients and controls adjusted for age, sex, and smoking status (pack-years) revealed that AMD was an independent variable associated with a lower RBC catalase (β = −0.37, p < 0.001) and higher PLT catalase (β = +0.25, p < 0.001), RBC GPx (β = +0.26, p < 0.001), PLT GPx (β = +0.16, p = 0.001), RBC R-GSSG (β = +0.13, p = 0.009), PLT R-GSSG (β = +0.12, p = 0.02) and RBC GSH transferase (β = +0.23, p < 0.001) activity. The activities of components of the antioxidant system were associated with disease severity and depended on dietary habits. The observed substantial increase in the activity of many critical endogenous antioxidants in patients with AMD further indicates that the required equilibrium in the antioxidant system is disturbed throughout the course of the disease. Our findings explicitly show that a diet rich in green vegetables, fish and omega-3-rich oils, supplemented by physical exercise, is beneficial for patients with AMD, as it might delay disease progression and help retain better visual function.

Oxidative stress parameters and antioxidants in patients with bipolar disorder: Results from a meta-analysis comparing patients, including stratification by polarity and euthymic status, with healthy controls.

To investigate oxidative stress markers and antioxidants in bipolar disorder (BD). Electronic MEDLINE/PubMed/Cochrane-Library/Scopus/TripDatabase search until 06/30/2019 for studies comparing antioxidant or oxidative stress markers between BD and healthy controls (HCs). Standardized mean differences (SMD) and 95% confidence intervals (CIs) were calculated for≥3 studies. Forty-four studies (n=3,767: BD=1,979; HCs=1,788) reported on oxidative stress markers malondialdehyde (MDA), thiobarbituric acid reactive substances (TBARS), and total nitrites; antioxidants glutathione (GSH), uric acid, and zinc; or antioxidantenhancing enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and GSH-transferase (GST). Compared with HCs, BD was associated with higher GST (P=.01), CAT (P=.02), nitrites (P<.0001), TBARS (P<.0001), MDA (P=.01), uric acid (P<.0001), and lower GSH (P=.006), without differences in SOD, GPX, and zinc. Compared to HCs, levels were higher in BD-mania for TBARS (P<.0001) and uric acid (P<.0001); in BD-depression for TBARS (P=.02); and BD-euthymia for uric acid (P=.03). Uric acid levels were higher in BD-mania vs BD-depression (P=.002), but not vs BD euthymia. TBARS did not differ between BD-mania and BD-depression. Medication-free BD-mania patients had higher SOD (P=.02) and lower GPX (P<.0001) than HCs. After treatment, BD did not differ from HCs regarding SOD and GPX. Beyond a single biomarker of oxidative stress, the combination of several parameters appears to be more informative for BD in general and taking into account illness polarity. BD is associated with an imbalance in oxidative stress with some phase-specificity for uric acid and TBARS and possible treatment benefits for SOD and GPX. Future studies should take into account confounding factors that can modify oxidative stress status and simultaneously measure oxidative stress markers and antioxidants including different blood sources.

Characterization of glutathione proteome in CHO cells and its relationship with productivity and cholesterol synthesis.

Glutathione (GSH) plays a central role in the redox balance maintenance in mammalian cells. Previous studies of industrial Chinese hamster ovary cell lines have demonstrated a relationship between GSH metabolism and clone productivity. However, a thorough investigation is required to understand this relationship and potentially highlight new targets for cell engineering. In this study, we have modulated the GSH intracellular content of an industrial cell line under bioprocess conditions to further elucidate the role of the GSH synthesis pathway. Two strategies were used: the variation of cystine supply and the direct inhibition of the GSH synthesis using buthionine sulfoximine (BSO). Over time of the bioprocess, a correlation between intracellular GSH and product titer has been observed. Analysis of metabolites uptake/secretion rates and proteome comparison between BSO‐treated cells and nontreated cells has highlighted a slowdown of the tricarboxylic acid cycle leading to a secretion of lactate and alanine in the extracellular environment. Moreover, an adaptation of the GSH‐related proteome has been observed with an upregulation of the regulatory subunit of glutamate–cysteine ligase and a downregulation of a specific GSH transferase subgroup, the Mu family. Surprisingly, the main impact of BSO treatment was observed on a global downregulation of the cholesterol synthesis pathways. As cholesterol is required for protein secretion, it could be the missing piece of the puzzle to finally elucidate the link between GSH synthesis and productivity.

Taenia solium glutathione transferase fraction activates macrophages and favors the development of Th1-type response.

Glutathione (GSH) transferase (GST) is an essential enzyme in cestodes for the detoxification of xenobiotics. In Taenia solium, two GSTs (Ts25GST and Ts26GST kDa) were isolated as a fraction (SGSTF) by GSH-Sepharose-4B. Both are located on the tegument. Immunization assays with SGSTF reduced up to 90% of the parasitic load in a murine model of cysticercosis. It prompted us to investigate how SGSTF induces this protective immune response. To test it, we exposed peritoneal macrophages to SGSTF for 24 h; such exposure favored the production of IL-12, TNF, and IL-10 as well as the expression of nitric oxide synthase 2 inducible (Nos2) and CD86, but did not induce the expression of chitinase-like 3 (Chil3). Confocal microscopy showed that the macrophages internalize the SGSTF which co-localized after 1 h with MHC-II in their plasma membranes. Macrophages exposed to SGSTF and co-cultured with anti-CD3 pre-activated T CD4+ cells, enhanced the proliferation of CD4+ cells, induced high interferon-γ (IFN-γ) secretion, and elevated the expression of CD25 and CD69, molecules associated with cell activation. Similar assay using T CD4+ cells from DO11.10 mice and ovalbumin (OVA) peptide+SGSTF as stimuli, showed enhanced cell proliferation and OVA-specific IFN-γ secretion. These data are in-line with those indicating that the P1, P5, and P6 peptides of Schistosoma japonicum 28GST highly promote T-cell proliferation and Th1 response in vitro. We found that such peptides are also present on Ts25GST and Ts26GST. It suggests that SGSTF activates peritoneal macrophages to a classically activated-like phenotype, and that these macrophages induce the differentiation of T CD4+ cells toward a Th1-type response.

Role of betaine in liver injury induced by the exposure to ionizing radiation.

Oxidative stress, apoptosis, and fibrosis may play a major role in the development of radiation-induced liver damage. Betaine, a native compound widely present in beetroot, was reported to possess hepato-protective properties. The objective of this study was to investigate the influence of betaine on radiation-induced liver damage. Animals were exposed to 9 Gy applied in 3 doses of 3 Gy/wk. Betaine (400 mg/kg/d), was orally supplemented to rats after the first radiation dose, and daily during the irradiation period. Animals were sacrificed 1 day after the last dose of radiation. The results showed that irradiation has induced oxidative stress in the liver denoted by a significant elevation in malondialdehyde, protein carbonyl, and 8-hydroxy-2-deoxyguanosine with a significant reduction in catalase activity and glutathione (GSH) content. The activity of the detoxification enzyme cytochrome P450 (CYP450) increased while GSH transferase (GSH-T) decreased. The activity of the apoptotic marker caspase-3 increased concomitant with increased hyaluronic acid, hydroxyproline, laminin (LN), and collagen IV. These alterations were associated with a significant increase of gamma-glutamyl transferase, alkaline phosphatase and alanine and aspartate aminotransferase markers of liver dysfunction. Betaine treatment has significantly attenuated oxidative stress, decreased the activity of CYP450, enhanced GSH-T, reduced the activity of caspase-3, and the level of fibrotic markers concomitant with a significant improvement of liver function. In conclusion, betaine through its antioxidant activity and by enhancing liver detoxification and reducing apoptosis may alleviate the progression of liver fibrosis and exert a beneficial impact on radiation-induced liver damage.

Activation versus inhibition of microsomal glutathione S-transferase activity by acrolein. Dependence on the concentration and time of acrolein exposure

The toxicity of acrolein, an α,β-unsaturated aldehyde, is due to its soft electrophilic nature and primarily involves the adduction of protein thiols. The thiol glutathione (GSH) forms the first line of defense against acrolein. The present study confirms that acrolein added to isolated rat liver microsomes can increase microsomal GSH transferase (MGST) activity 2–3 fold, which can be seen as a direct adaptive increase in the protection against acrolein. At a relatively high exposure level, acrolein appeared to inhibit MGST. The activation is due to adduction of thiol groups, and the inactivation probably involves adduction of amino groups in the enzyme by acrolein. The preference of acrolein to react with thiol groups over amino groups can explain why the enzyme is activated at a low exposure level and inhibited at a high exposure level of acrolein. These opposite forms of direct adaptation on the level of enzyme activity further narrow the thin line between survival and promotion of cell death, governed by the level of exposure.

Crystal structure of yeast monothiol glutaredoxin Grx6 in complex with a glutathione-coordinated [2Fe-2S] cluster.

Glutaredoxins (Grxs) constitute a superfamily of proteins that perform diverse biological functions. The Saccharomyces cerevisiae glutaredoxin Grx6 not only serves as a glutathione (GSH)-dependent oxidoreductase and as a GSH transferase, but also as an essential [2Fe-2S]-binding protein. Here, the dimeric structure of the C-terminal domain of Grx6 (holo Grx6C), bridged by one [2Fe-2S] cluster coordinated by the active-site Cys136 and two external GSH molecules, is reported. Structural comparison combined with multiple-sequence alignment demonstrated that holo Grx6C is similar to the [2Fe-2S] cluster-incorporated dithiol Grxs, which share a highly conserved [2Fe-2S] cluster-binding pattern and dimeric conformation that is distinct from the previously identified [2Fe-2S] cluster-ligated monothiol Grxs.

Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola.

BackgroundGlutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection.ResultsMining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were ‘orphans’. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues.ConclusionsAmong the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0462-0) contains supplementary material, which is available to authorized users.

Early Life Lung Antioxidant Levels and Response to Ozone: Influence of Sex and Maturation in Wistar Rats

Epidemiologic studies of air pollution effects on respiratory health report significant modification by sex. Studies of children suggest stronger effects among boys in early life and girls in later childhood. In adults, particularly the elderly, studies report stronger effects among women. Differential inflammatory or antioxidant responses are purported. Herein we examined sex or maturation influences in prepubertal Wistar rats [a strain prone to ozone (O3)‐induced lung injury/inflammation] on (A) lung antioxidant and related enzyme levels and (B) antioxidant response to O3. Female (F) and male (M) 14, 21, and 28 d (pre‐, at, post‐weaning) pups were exposed to air, 0.5 or 1 ppm O3 x 2h. In air controls, no sex‐based differences in lung antioxidant levels were observed. From 14‐28 d, weight increased (F: 1.7‐2.7‐fold; M: 1.7‐3.1‐fold); with M&gt;F at 28 d. Likewise, lung uric acid increased (1.1‐1.2‐fold), as did ascorbic acid (1.4‐2.7‐fold), glutathione (GSH) peroxidase (1.0‐3.5‐fold), GSH transferase (1.0‐2.9‐fold), GSH reductase (1.2‐4.2‐fold), and G‐6‐PDH (10‐fold) (per gwet wt). Conversely, SOD declined 30‐42%. Lung GSH was relatively stable. At 0h, 1 ppm O3‐exposed F 14 d pups had decreased GSH and related enzyme activity. At 21 d, exposure was without effect on GSH; and males showed O3‐induced increases in uric acid. At 28 d, no antioxidant changes were apparent. Together, data suggest health risk due to early life pollutant exposure is greatest in very immature females. With maturity, especially post‐weaning, rat lung antioxidant reserve increases, thus reducing susceptibility to pollutant‐induced oxidative stress. (Abstract does not reflect USEPA policy)

Regulation of Glutathione in Health and Disease with Special Emphasis on Chronic Alcoholism and Hyperglycaemia Mediated Liver Injury: A Brief Perspective

Glutathione (GSH) plays a major role in the cell due to its antioxidant properties. The transcription factor Nrf2 plays an important role in the synthesis of GSH in the cell. Increased cellular GSH content leads to greatly decreased oxidative stress and toxicity. Alcoholism and hyperglycaemia both lead to liver injury, and GSH plays a crucial role in ameliorating the hepatic damage under these pathophysiological conditions. Further, GSH transferase which is involved in drug detoxification through conjugation of xenobiotics with GSH exhibits polymorphic forms which affect its detoxification efficacy. Further, GSH supplementation through NAC and UDCA and GSH depletion through BSO and DEM lessen and aggravate cellular dysfunction, respectively. Therefore, GSH regulation which occurs through several mechanisms has a very important role in maintaining or disrupting the oxidative environment of the cell.

Mutation Spectra of S-(2-Hydroxy-3,4-epoxybutyl)glutathione: Comparison with 1,3-Butadiene and Its Metabolites in the Escherichia coli rpoB Gene

S-(2-Hydroxy-3,4-epoxybutyl)glutathione (DEB-GSH conjugate) is formed from the reaction of 1,2:3,4-diepoxybutane (DEB) with glutathione (GSH), and the conjugate is considerably more mutagenic than several other butadiene-derived epoxides-including DEB-in Salmonella typhimurium TA1535 [Cho, S.-H., (2010) Chem. Res. Toxicol. 23, 1544-1546]. We previously identified six DNA adducts in the reaction of the DEB-GSH conjugate with nucleosides and calf thymus DNA and two DNA adducts in livers of mice and rats treated with DEB [Cho, S.-H. and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 706-712]. To define the role of GSH conjugation in 1,3-butadiene (BD) metabolism and characterize the mechanism of GSH transferase (GST)-enhanced mutagenicity of DEB, mutation spectra of BD and its metabolites in the absence and presence of GST/GSH and mouse liver microsomes were compared in the rpoB gene of Escherichia coli TRG8. The presence of GST considerably enhanced mutations. The mutation spectra derived from the DEB-GSH conjugate, the DEB/GST/GSH system, and the BD/mouse liver microsomes/GST/GSH system matched each other and were different from those derived from the other systems devoid of GSH. The major adducts in E. coli TRG8 cells treated with the DEB/GST/GSH system, the BD/mouse liver microsomes/GST/GSH system, or the DEB-GSH conjugate were S-[4-(N(7)-guanyl)-2,3-dihydroxybutyl]GSH, S-[4-(N(3)-adenyl)-2,3-dihydroxybutyl]GSH, and S-[4-(N(6)-deoxyadenosinyl)-2,3-dihydroxybutyl]GSH, indicating the presence of the GSH-containing DNA adducts in the systems. These results, along with the strong enhancement of mutagenicity by GST in this system, indicate the relevance of these GSH-containing DNA adducts.

Effects of sublethal, environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis

Hexavalent chromium Cr(VI) is an important contaminant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. In the marine bivalve Mytilus galloprovincialis exposure to non-toxic, environmentally relevant concentrations of Cr(VI) was shown to modulate functional parameters and gene expression in both the digestive gland and hemocytes. In this work, the effects of exposure to Cr(VI) (0.1–1–10μgL−1animal−1 for 96h) in mussel gills were investigated. Gill morphology and immunolocalization of GSH-transferase (GST), of components involved in cholinergic (AChE and ChAT), adrenergic (TH) and serotoninergic (5-HT3 receptor) systems, regulating gill motility, were evaluated. Total glutathione content, activities of GSH-related enzymes (glutathione reductase – GSR, GST), of catalase, and of key glycolytic enzymes (phosphofructokinase – PFK and pyruvate kinase – PK) were determined. Moreover, mRNA expression of selected Mytilus genes (GST-π, metallothionein isoforms MT10 and MT20, HSP70 and 5-HT receptor) was assessed by RT-q-PCR. Cr(VI) exposure induced progressive changes in gill morphology and in immunoreactivity to components involved in neurotransmission that were particularly evident at the highest concentration tested, and associated with large metal accumulation. Cr(VI) increased the activities of GST and GSR, and total glutathione content to a different extent at different metal concentrations, this suggesting Cr(VI) detoxication/reduction at the site of metal entry. Cr(VI) exposure also increased the activity of glycolytic enzymes, indicating modulation of carbohydrate metabolism. Significant changes in transcription of different genes were observed. In particular, the mRNA level for the 5-HTR was increased, whereas both decreases and increases were observed for GST-π, MT10, MT20 and HSP70 mRNAs, showing sex- and concentration-related differences. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in mussel gills, and indicate that this tissue represents the major target of exposure to environmentally relevant concentrations of the metal.

Conjugation of Butadiene Diepoxide with Glutathione Yields DNA Adducts in Vitro and in Vivo

1,2,3,4-Diepoxybutane (DEB) is reported to be the most potent mutagenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant. DEB is capable of inducing the formation of monoalkylated DNA adducts and DNA-DNA and DNA-protein cross-links. We previously reported that DEB forms a conjugate with glutathione (GSH) and that the conjugate is considerably more mutagenic than several other butadiene-derived epoxides, including DEB, in the base pair tester strain Salmonella typhimurium TA1535 [Cho et al. (2010) Chem. Res. Toxicol. 23, 1544-1546]. In the present study, we determined steady-state kinetic parameters of the conjugation of the three DEB stereoisomers-R,R, S,S, and meso (all formed by butadiene oxidation)-with GSH by six GSH transferases. Only small differences (<3-fold) were found in the catalytic efficiency of conjugate formation (k(cat)/K(m)) with all three DEB stereoisomers and the six GSH transferases. The three stereochemical DEB-GSH conjugates had similar mutagenicity. Six DNA adducts (N(3)-adenyl, N(6)-adenyl, N(7)-guanyl, N(1)-guanyl, N(4)-cytidyl, and N(3)-thymidyl) were identified in the reactions of DEB-GSH conjugate with nucleosides and calf thymus DNA using LC-MS and UV and NMR spectroscopy. N(6)-Adenyl and N(7)-guanyl GSH adducts were identified and quantitated in vivo in the livers of mice and rats treated with DEB ip. These results indicate that such DNA adducts are formed from the DEB-GSH conjugate, are mutagenic regardless of sterochemistry, and are therefore expected to contribute to the carcinogenicity of DEB.

Interaction of Nefopam and Orphenadrine with the Cytochrome P-450 and the Glutathione System in Rat Liver

Nefopam, a cyclic analogue of orphenadrine, exhibits a type I (substrate) and a type II (ligand) interaction with ferri-cytochrome P-450 in control and phenobarbitone induced rat hepatic microsomes respectively. In-vitro metabolism of nefopam in phenobarbitone-induced microsomes leads to the production of a reactive metabolite which complexes with cytochrome P-450. In contrast to the known complexation of orphenadrine, complexation by nefopam can be inhibited by glutathione (GSH, 0.1-1.0 mM). However, in-vivo administration of nefopam to rats does not diminish the GSH content of liver cytosol nor increase oxidized glutathione levels nor alter the activities of GSH transferase and GSH peroxidase. In-vivo administration does not lead to cytochrome P-450 induction nor cytochrome P-450 complexation as has been shown for orphenadrine. Finally, nefopam inhibits the NADPH dependent endogenous H2O2 production in both control and phenobarbitone-induced microsomes.

Glutathione Transferase P1 Interacts Strongly with the Inner Leaflet of the Plasma Membrane

GSH transferases (GSTs) are a superfamily of proteins best known for detoxifying harmful electrophilic compounds by catalyzing their conjugation with GSH. GSTP1 is the most prevalent and widely distributed GST in human tissues, helping to detoxify a diverse array of carcinogens and drugs. In contrast with its protective role, overexpression of GSTP1 in a variety of malignancies is associated with a poor prognosis due to failure of chemotherapy. Although GSTP1 is classified as a cytosolic GST, we discovered previously that it is associated with the plasma membrane of the small cell lung cancer cell lines, H69 and H69AR. In the current study, endogenous and overexpressed GSTP1 in human embryonic kidney (HEK) 293 and MCF-7 cell lines, respectively, were found also to associate with the plasma membrane, indicating that this interaction is not unique to H69 and H69AR cells. GSTP1 immunostaining in HEK293 and MCF7-GSTP1 cells only occurred under permeabilized conditions, suggesting that GSTP1 is associated with the intracellular surface of the plasma membrane. Cell surface biotinylation studies confirmed this finding. Immunogold electron microscopy revealed the presence of GSTP1 in close proximity to the plasma membrane. GSTP1 was not dissociated from plasma membrane sheets by high salt [potassium iodide (KI; 1 M) or KI/EDTA (1 M/2 mM)] or alkaline Na(2)CO(3) (100 mM, pH 11.4), conditions known to strip peripherally associated membrane proteins. Thus, we report for the first time that GSTP1 is associated with the inner leaflet of the plasma membrane through a remarkably strong interaction.

Pleiotropic effects of hexavalent chromium (CrVI) in Mytilus galloprovincialis digestive gland

Hexavalent Chromium Cr(VI) is an important contaminant considered as a model oxidative toxicant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. On the other hand, in mammals the reduced form Cr(III) is considered an essential microelement, involved in regulation of lipid and carbohydrate metabolism; moreover, recent evidence suggests that Cr may have endocrine effects. In this work, the effects of Cr(VI) were investigated in the digestive gland of the marine bivalve Mytilus galloprovincialis. Mussels were exposed to 0.1–1–10–100 μg Cr(VI) L −1 animal −1 for 96 h. At 100 μg L −1, a large increase in total Cr tissue content was observed; in these conditions, the lysosomal membranes were completely destabilized, whereas other lysosomal biomarkers (neutral lipids-NL and lipofuscin-LF), as well as different enzyme activities and gene expression were unaffected, this indicating severe stress conditions in the tissue. On the other hand, at lower concentrations, changes in other histochemical, biochemical and molecular endpoints were observed. In particular, at both 1 and 10 μg L −1, lysosomal destabilization was associated with significant NL and LF accumulation; however, no changes in catalase and GSH transferase (GST) activities were observed. At the same concentrations, GSSG reductase (GSR) activity was significantly increased, this probably reflecting the recycling of GSSG produced in the GSH-mediated intracellular reduction of Cr(VI). Increased activities of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase) were also observed, indicating that Cr(VI) could affect carbohydrate metabolism. Cr(VI) induced downregulation or no effects on the expression of metallothioneins MT10 and MT20, except for an increase in MT20 transcription in males. Moreover, significant up-regulation of the Mytilus estrogen receptor MeER2 and serotonin receptor (5-HTR) were observed in both sexes. The results demonstrate that exposure to Cr(VI) in the low ppb range did not result in strong toxicity or oxidative stress conditions in mussel digestive gland. On the other hand, our data support the hypothesis that low concentrations of the metal can exert pleiotropic effects on mussel physiology, from modulation of lipid and carbohydrate metabolism, to effects on the expression of estrogen-responsive genes.

Structure and Function of YghU, a Nu-Class Glutathione Transferase Related to YfcG from Escherichia coli

The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (K(d1) = 0.07 ± 0.03 mM) and one weak (K(d2) = 1.3 ± 0.2 mM) binding site for GSH. YghU exhibits little or no GSH transferase activity with most typical electrophilic substrates but does possess a modest catalytic activity toward several organic hydroperoxides. Most notably, the enzyme also exhibits disulfide-bond reductase activity toward 2-hydroxyethyl disulfide [k(cat) = 74 ± 6 s(-1), and k(cat)/K(M)(GSH) = (6.6 ± 1.3) × 10(4) M(-1) s(-1)] that is comparable to that previously determined for YfcG. A superposition of the structures of the YghU·2GSH and YfcG·GSSG complexes reveals a remarkable structural similarity of the active sites and the 2GSH and GSSG molecules in each. We conclude that the two structures represent reduced and oxidized forms of GSH-dependent disulfide-bond oxidoreductases that are distantly related to glutaredoxin 2. The structures and properties of YghU and YfcG indicate that they are members of the same, but previously unidentified, subfamily of GSH transferase homologues, which we suggest be called the nu-class GSH transferases.

Arginine 104 Is a Key Catalytic Residue in Leukotriene C4 Synthase

Human leukotriene C4 synthase (hLTC4S) is an integral membrane enzyme that conjugates leukotriene (LT) A4 with glutathione to form LTC4, a precursor to the cysteinyl leukotrienes (LTC4, LTD4, and LTE4) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC4S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3–99.9% of the specific activity against LTA4. Steady-state kinetics of R104A and R104S revealed that the Km for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λmax at 239 nm, could be detected using R104A or R104S hLTC4S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pKa to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA4. On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC4S by 88 and 70%, respectively, without significantly affecting the kcat/Km values for GSH, and a crystal structure of R31Q hLTC4S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC4S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.

Characterization of the hydrophobic substrate-binding site of the bacterial beta class glutathione transferase from Proteus mirabilis

Since their discovery, bacterial glutathione (GSH)transferases have been characterized in terms of their ability to catalyse a variety of different reactions on a large set of toxic molecules of xenobiotic or endobiotic origin. Furthermore the contribution of different residues in the GSH-binding site to GSH activation has been extensively investigated. Little is known, however, about the contribution to catalysis and overall stability of single residues shaping the hydrophobic co-substrate binding site (H-site). Here we tackle this problem by site-directed mutagenesis of residues facing the H-site in the bacterial beta class GSH transferase from Proteus mirabilis. We investigate the behaviour of these mutants under a variety of conditions and analyse their activity against several co-substrates, representative of the different reactions catalyzed by bacterial GSH transferases. Our work shows that mutations at the H-site can be used to modulate activity at the level of the different catalytic mechanisms operating on the chosen substrates, each mutation showing a different fingerprint. This work paves the way for future studies aimed at improving the catalytic properties of beta class GSH transferases against selected substrates for bioremediation purposes.