Structural and Functional Characterization of Camelus dromedarius Glutathione Transferase M1-1

Glutathione transferases (GSTs; EC. 2.5.1.18) are a large family of multifunctional enzymes that play crucial roles in the metabolism and inactivation of a broad range of xenobiotic compounds. In the present work, we report the kinetic and structural characterization of the isoenzyme GSTM1-1 from Camelus dromedarius (CdGSTM1-1). The CdGSΤM1-1 was expressed in E. coli BL21 (DE3) and was purified by affinity chromatography. Kinetics analysis showed that the enzyme displays a relative narrow substrate specificity and restricted ability to bind xenobiotic compounds. The crystal structures of CdGSΤM1-1 were determined by X-ray crystallography in complex with the substrate (GSH) or the reaction product (S-p-nitrobenzyl-GSH), providing snapshots of the induced-fit catalytic mechanism. The thermodynamic stability of CdGSTM1-1 was investigated using differential scanning fluorimetry (DSF) in the absence and in presence of GSH and S-p-nitrobenzyl-GSH and revealed that the enzyme’s structure is significantly stabilized by its ligands. The results of the present study advance the understanding of camelid GST detoxification mechanisms and their contribution to abiotic stress adaptation in harsh desert conditions.

The relationship of polymorphic variants of genes GSTM1, GSTT1, GSTP1 with the risk of developing diseases (literature review)

Authors analyzed the relationship of the most studied polymorphisms of glutathione-S-transferase genes (GSTT1, GSTM1, GSTP1) with the risk of developing diseases in the territory of the Russian Federation. The authors analyzed domestic articles on gene polymorphisms and their association with various conditions. A systematic review of scientific papers was carried out using the following electronic databases: Cyberleninka, Electronic Library and Google Scholar, which searched for glutathione transferase (GST) gene polymorphisms from 1900 to 2020. The following keywords were used for the search: “GST polymorphism”. The search for articles was carried out in English but took into account the results obtained only in the territory of the Russian Federation. The search for scientific publications was independently checked and compared to filter out duplicate articles. The sample size, the number of loci included in the analysis, and the analyzed population (ethnic group) are essential in studying the relationship between glutathione S-transferase gene polymorphisms and the development of diseases. In general, the analysis of the articles makes it possible to reveal the association between the polymorphisms of the glutathione-S-transferase genes and the high risk of developing oncological diseases, respiratory diseases and other pathologies. This article did not consider the relationship of polymorphisms with reproductive pathologies because this topic includes a large amount of work and requires separate consideration. The least studied issue is the relationship between polymorphisms of genes for biotransformation of xenobiotics and the development of occupational diseases.

The Effect of Fullerenol C60(OH)36 on the Antioxidant Defense System in Erythrocytes

Background: Fullerenols (water-soluble derivatives of fullerenes), such as C60(OH)36, are biocompatible molecules with a high ability to scavenge reactive oxygen species (ROS), but the mechanism of their antioxidant action and cooperation with endogenous redox machinery remains unrecognized. Fullerenols rapidly distribute through blood cells; therefore, we investigated the effect of C60(OH)36 on the antioxidant defense system in erythrocytes during their prolonged incubation. Methods: Human erythrocytes were treated with fullerenol at concentrations of 50–150 µg/mL, incubated for 3 and 48 h at 37 °C, and then hemolyzed. The level of oxidative stress was determined by examining the level of thiol groups, the activity of antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, and glutathione transferase), and by measuring erythrocyte microviscosity. Results: The level of thiol groups in stored erythrocytes decreased; however, in the presence of higher concentrations of C60(OH)36 (100 and 150 µg/mL), the level of -SH groups increased compared to the control. Extending the incubation to 48 h caused a decrease in antioxidant enzyme activity, but the addition of fullerenol, especially at higher concentrations (100–150 µg/mL), increased its activity. We observed that C60(OH)36 had no effect on the microviscosity of the interior of the erythrocytes. Conclusions: In conclusion, our results indicated that water-soluble C60(OH)36 has antioxidant potential and efficiently supports the enzymatic antioxidant system within the cell. These effects are probably related to the direct interaction of C60(OH)36 with the enzyme that causes its structural changes.

Design of a graphene oxide-BODIPY conjugate for glutathione depletion and photodynamic therapy

Boron dipyrromethene derivates (BODIPYs) are promising photosensitisers (PSs) for cancer treatment using photodynamic therapy (PDT). This study investigates the functionalisation of graphene oxide (GO) with a BODIPY derivate for glutathione (GSH) depletion and PDT. The functionalisation of GO with a 3,5-dichloro-8-(4-boronophenyl) BODIPY via a diol derivatisation with the phenyl boronic acid moiety at the meso position of the BODIPY core, allowed to preserve the intrinsic properties of GO. We demonstrated that both chlorine atoms were substituted by GSH in the presence of glutathione transferase (GST), inducing a relevant bathochromic shift in the absorption/emission features and thus generating the active PS. Ex vitro assessment using cell lysates containing cytoplasmatic GST revealed the intracellular catalytic mechanism for the nucleophilic substitution of the GO-BODIPY adduct with GSH. Confocal microscopy studies showed important differences in the cellular uptake of free BODIPY and GO-BODIPY and revealed the coexistence of GO-BODIPY, GO-BODIPY-GS, and GO-BODIPY-GS2 species inside vesicles and in the cytoplasm of the cells after 24 h of incubation. In vitro biocompatibility and safety of GO and GO-BODIPY were evaluated in 2D and 3D models of prostate adenocarcinoma cells (PC-3), where no toxicity was observed up to 100 µg/mL of GO/GO-BODIPY in all treated groups 24 h post-treatment (cell viability > 90%). Only a slight decrease to 80% at 100 µg/mL was observed after 48 h of incubation. We demonstrated the efficacy of a GO adduct containing an α-chlorine-substituted BODIPY for the simultaneous depletion of intracellular GSH and the photogeneration of reactive oxygen species using a halogen white light source (5.4 mW/cm2) with a maximum in the range of 500-800 nm, which significantly reduced cell viability (< 50%) after irradiation. Our study provides a new vision on how to apply BODIPY derivates and potentiate the toxicity of PDT in prostate and other types of cancer.

Oxidative stress and antioxidant enzyme levelsin the blood serum of white mice when exposed to iron and manganese in drinking water under experimental conditions

The object of the study of enzymatic antioxidants in the blood serum of white mice were 30 outbred white male and female mice of the same age and approximately of the same weight. The animals were divided into 3 groups: one control group and two experimental ones in which the mice consumed water with a high content of total iron and manganese (II) ion (over 5 MPC).This study revealed that the activity of glutathione transferase as a marker of antioxidant defense increased among the male mice in the experimental group after experiencing a 12-week-long intoxication, however the level of malondialdehyde in their blood serum indicated insignificant oxidative stress. Keywords: iron, manganese, drinking water, glutathione transferase, malondialdehyde, antioxidant defense, oxidative stress.

Metabolic and Redox Biomarkers in Skeletal Muscle Underlie Physiological Adaptations of Two Estivating Anuran Species in a South American Semi-arid Environment

The upregulation of endogenous antioxidants (i.e., preparation for oxidative stress, POS) is part of the biochemical responses underlying the adaptation of animals to adverse environments. Despite the phylogenetic diversity of animals in which POS has been described, most studies focus on animals under controlled laboratory conditions. To address this limitation, we have recently assessed the redox metabolism in the skeletal muscle of Proceratophrys cristiceps estivating under natural settings in the Caatinga. Here, we analyzed biochemical biomarkers in the muscle of another Caatinga species, Pleurodema diplolister, during the rainy (active) and dry (estivating frogs) seasons. We aimed to determine whether P. diplolister enhances its antioxidants during estivation under field conditions and to identify any effect of species on the biochemical responses of P. diplolister and P. cristiceps associated with estivation. To do so, we measured the activities of representative enzymes of intermediary metabolism and antioxidant systems, as well as glutathione and protein carbonyl levels, in the skeletal muscle of P. diplolister. Our findings revealed the suppression of oxidative metabolism and activation of antioxidant enzymes in estivating P. diplolister compared with active specimens. No changes in oxidative damage to proteins were observed and estivating P. diplolister had lower levels of disulfide glutathione (GSSG) and disulfide-to-total glutathione ratio (GSSG/tGSH) than those observed in active individuals. When data for P. diplolister and P. cristiceps were assembled and analyzed, significant effects of species were detected on the activities of metabolic enzymes (citrate synthase, isocitric dehydrogenase, malic enzyme, and creatine kinase) and antioxidant enzymes (catalase, glutathione peroxidase and glutathione transferase), as well as on GSSG/tGSH ratio. Such effects might underlie the physiological and behavioral differences between these two species that share the same microhabitat and survival strategy (i.e., to estivate) during the dry season. Despite some peculiarities, which reflect the physiological diversity of the mechanisms associated with estivation in the Brazilian Caatinga, both P. diplolister and P. cristiceps seem to balance the suppression of oxidative pathways, the maintenance of the capacity of oxygen-independent pathways, and the activation of endogenous antioxidants to preserve muscle function and be ready to resume activity whenever the unpredictable rainy period arrives.

Molecular Characterization of Prdx Family and Response of Antioxidant Enzymes in Berberine Hydrochloride-Treated Charybdis japonica Infected With Aeromonas hydrophila

Berberine hydrochloride is an isoquinoline alkaloid, which has antitumoral, antibacterial, and antiviral activities in vivo and in vitro. Charybdis japonica is one of the main economic species of crab in Southeast Asia. We studied the molecular mechanism of oxidative stress in berberine hydrochloride-treated C. japonica infected with Aeromonas hydrophila. C. japonica were infected with A. hydrophila after being submerged in different concentrations (0, 100, 200, and 300 mg/L) of berberine hydrochloride for 48 h. The full-length cDNA of Prx6 and the ORFs of Prx5 and PXL2A were cloned. Prx6 and PXL2A each have one conserved domain, Cys44, and Cys81. The Prx5 conserved domain contains three important Cys loci, Cys75, Cys100, and Cys76. Prx6 was different from Prx5 and PXL2A in the Peroxiredoxin family. The transcription levels of PXL2A infected with A. hydrophila were all higher than the control. The transcription levels of C. japonica were further increased by adding berberine hydrochloride and were increased the highest at a concentration of 300 mg/L. The activities of glutathione peroxidase, superoxide dismutase, and catalase in the hepatopancreas of berberine hydrochloride-treated C. japonica infected with A. hydrophila were significantly increased compared with those only infected with A. hydrophila and the control group. The glutathione transferase activity in the hepatopancreas was significantly increased in berberine hydrochloride-treated C. japonica. The results of this study provide a new understanding of the potential role of berberine hydrochloride on the oxidative stress mechanisms of C. japonica.

Glutathione-dependent system in the blood of gastric cancer patients with various tumor histotypes and prevalence of the disease

Purpose of the study. Exploring the erythrocyte glutathione system functioning in patients with gastric cancer in a comparative aspect, depending on tumor histotype and disease prevalence.Patients and methods. The study included 89 patients with gastric cancer, divided into 6 groups depending on the histotype of the tumor. Separately, the results of the study were analyzed in patients with T4 status according to TNM and in patients at stage 4. The content of reduced glutathione and the activity of glutathione-dependent enzymes in the erythrocytes of the patients' blood were studied by conventional spectrophotometric methods. Statistical processing of the results was carried out using the Statistika 6.0 software package according to the Student's t-test and the nonparametric Wilcoxon-Mann-Whitney test for two independent samples.Results. An increase in the content of glutathione was revealed in patients with gastric cancer compared with the group without oncopathology. The maximum increase was observed in patients with low-grade adenocarcinoma - by 42.5 %, while in signet ring cell carcinoma (SRCC) there was only a tendency to increase by 17.8 %. Glutathione reductase activity was decreased in adenocarcinoma by 23.4-26.2 % and did not change in SRCC. The activity of the antioxidant enzymes glutathione peroxidase and glutathione transferase was increased in all groups, and especially in SRCC - by 76 % and 23-29 %, respectively. In patients with the T4 status and at stage IV of the process, a lower activity of the studied glutathione-dependent enzymes was revealed in comparison with all other groups of patients.Conclusions. The data obtained indicate a greater functional potential of the glutathione system in SRCC. A significant increase in glutathione transferase activity at a sufficiently high level of reduced glutathione can contribute to the development of treatment resistance in SRCC patients.

Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Preparation and characterization of recombinant human glutathione transferase P1 and screening of novel enzyme inhibitors

Human glutathione transferase P1 (GSTP1) plays an important role in the second phase of xenobiotic biotransformation and in the regulation of apoptotic signal pathways. Directed screening of new enzyme inhibitors is an actual task since selective suppression of GSTP1 activity in tumor cells may substantially increase their sensitivity to chemotherapy. Known methods to obtain recombinant GSTP1 with a hexahistidine tag in the structure are complex, laborious, and suffer from significant losses of the enzyme activity. With the aim to create a simple and effective bacterial expression system for tagless GSTP1 posessing native structure and high activity, in the present work the full-length gstp1 gene was cloned into the pTXB1 plasmid vector, followed by transformation of E. coli cells. The optimized expression level amounted to 30–32 mg of the enzyme per liter of broth. Using glutathione-containing affinity membranes, the purified enzyme was isolated from bacterial lysate with the yield of 75.7 % and specific activity of 102.6 U/mg protein. The enzyme homogeneity was confirmed by gel-electrophoretic and mass-spectrometric data. Physico-chemical and catalytic properties of recombinant GSTP1 practically coincided with those of the native erythrocytary enzyme. The results of in silico and in vitro screening allowed to reveal structural factors and interactions determining the efficiency of the enzyme inhibition by carbocyclic and N-heterocyclic ligands. The preferable orientation of “good” inhibitors in the GSTP1 H-site was also established. Three strong enzyme inhibitors were found: 1,10-phenanthroline-5,6-dione, Alizarin Red S, and indigo carmine, with their respective IC50 values of 31, 16 and 2.3 μM. The new inhibitors are of certain interest for the development of novel lead structures with potential antitumor activity.