Pi-class Glutathione S-transferase Research Articles

Virtual screening, MMGBSA, and molecular dynamics approaches for identification of natural products from South African biodiversity as potential Onchocerca volvulus pi-class glutathione S-transferase inhibitors

We investigated 1012 molecules from natural products previously isolated from the South African biodiversity (SANCDB, https://sancdb.rubi.ru.ac.za/), for putative inhibition of Onchocerca volvulus pi-class glutathione S-transferase (Ov-GST2) by virtual screening, MMGBSA, and molecular dynamics approaches. ADMET, docking, and MMGBSA shortlisted 12 selected homoisoflavanones-type hit molecules, among which two namely SANC00569, and SANC00689 displayed high binding affinities of −46.09 and −46.26 kcal mol−1, respectively towards π-class Ov-GST2, respectively. The molecular dynamics results of SANC00569 showed the presence of intermolecular H-bonding, hydrophobic interactions between the ligand and key amino acids of Ov-GST2, throughout the simulation period. This hit molecule had a stable binding pose and occupied the binding pockets throughout the 200 ns simulation. To the best of our knowledge, there is no report of any alleged anti–onchocerciasis activity referring to homoisoflavanones or flavonoids. Nevertheless, homoisoflavanones, which are a subclass of flavonoids, exhibit a plethora of biological activities. All these results led to the conclusion that SANC00569 is the most hypothetical Ov-GST2, which could lead the development of new drugs against Onchocerca volvulus pi-class glutathione S-transferase. Further validation of these findings through in vitro and in vivo studies is required.

Cell Dome as an Evaluation Platform for Organized HepG2 Cells.

Human-hepatoblastoma-derived cell line, HepG2, has been widely used in liver and liver cancer studies. HepG2 spheroids produced in a three-dimensional (3D) culture system provide a better biological model than cells cultured in a two-dimensional (2D) culture system. Since cells at the center of spheroids exhibit specific behaviors attributed to hypoxic conditions, a 3D cell culture system that allows the observation of such cells using conventional optical or fluorescence microscopes would be useful. In this study, HepG2 cells were cultured in "Cell Dome", a micro-dome in which cells are enclosed in a cavity consisting of a hemispherical hydrogel shell. HepG2 cells formed hemispherical cell aggregates which filled the cavity of Cell Domes on 18 days of culture and the cells could continue to be cultured for 29 days. The cells at the center of hemispherical cell aggregates were observed using a fluorescence microscope. The cells grew in Cell Domes for 18 days exhibited higher Pi-class Glutathione S-Transferase enzymatic activity, hypoxia inducible factor-1α gene expression, and higher tolerance to mitomycin C than those cultured in 2D on tissue culture dishes (* p < 0.05). These results indicate that the center of the glass adhesive surface of hemispherical cell aggregates which is expected to have the similar environment as the center of the spheroids can be directly observed through glass plates. In conclusion, Cell Dome would be useful as an evaluation platform for organized HepG2 cells.

Improving reactivity of naphthalimide-based GST probe by imparting TPP cation: Development and application for live cell imaging

Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes comprising multiple classes and subtypes. This paper describes the synthesis and characterization of TPPBN-1, a naphthalimide derivative conjugated with a triphenylphosphonium (TPP) cation. When 4-bromonaphthalimide (BrNaph), a previously characterized GST substrate, was conjugated to a TPP cation, the conjugate showed increased reactivity towards most alpha- and mu-class GSTs, particularly the GSTA2 subtype, compared to the parent compound, but hardly towards Pi-class GSTs. Using this probe with enhanced reactivity, the enzymatic activity of endogenous GSTA1/2 in HepG2 cells was visualized by confocal fluorescence microscopy. The results demonstrated that modification with TPP cations, which are often used as tags for targeting mitochondria, can be used to enhance the reactivity of probes for specific GST subtypes.

Alexander Munn

An immunohistochemical study of glutathione S-transferase (GST) expression in hepatocellular carcinoma and cholangiocarcinoma is described. Unlike most animal models of hepatic malignancy pi class GST was not consistently overexpressed in...

Pi-Class Glutathione S-transferase (GSTP1)-Selective Fluorescent Probes for Multicolour Imaging with Various Cancer-Associated Enzymes.

Pi-class glutathione S-transferase (GSTP1) is highly expressed in a wide variety of human cancer tissues compared to the corresponding normal counterpart. Therefore, GSTP1 is a potential target enzyme for overcoming resistance to chemotherapeutic agents or visualizing specific lesions such as cancer. Here, we present orange and red fluorescence-emitting probes selective for GSTP1. Carbofluorescein and TokyoMagenta fluorophores were modified with a previously described GSTP1-selective chromogenic compound to generate orange and red fluorescence probes, respectively. Of these probes, Ps-CF, the orange fluorescence-emitting probe, was confirmed to be highly specific for detecting GSTP1 exogenously or endogenously expressed in various cancer cells. Additionally, it was demonstrated that Ps-CF is applicable for the simultaneous detection of GSTP1 and another cancer-associated enzyme by using a green fluorescence emitting γ-glutamyl transpeptidase (GGT) probe. In conclusion, the fluorescent probes developed in this study enable the simultaneous detection of multiple tumour markers such as GSTP1 with other cancer-associated enzymes by concurrently using spectrally distinguished fluorescent probes, potentially broadening the scope of cancer detection.

Design and synthesis of versatile GSTP1-specific fluorogenic substrates for the highly sensitive detection of GSTP1 activity in living cells

ABSTRACT: Pi-class glutathione S-transferase (GSTP1) is a detoxification enzyme that is highly expressed in various types of cancer cells and is a promising target for cancer imaging and therapy. Ps-TAc, an acetylated derivative of the GSTP1-specific fluorogenic substrate Ps-TG, is attracting attention as an effective GSTP1 fluorescent probe, and has been successfully used to visualize intracellular GSTP1 activity. Ps-TAc is a prodrug type fluorescent probe in which the phenolic hydroxyl group of Ps-TG is acetylated and thus is susceptible to nonspecific hydrolysis, potentially compromising its ability to detect GSTP1 activity. Here, we describe the development of a highly selective fluorogenic GSTP1 substrate that is membrane permeable and does not involve esterification and show its application to live-cell imaging and FACS analysis. We designed and synthesized several compounds with benzylsulfone substituents instead of the mesyl group of Ps-TG and tested their fluorescence activation by GSTP1 catalysis in vitro and in cellulo. Of the test compounds, Ps-TG3 was the most suitable for the visualization of intracellular GSTP1 activity because the signal from living cells increased significantly when MK-571, an inhibitor of multidrug resistance proteins (MRPs), was simultaneously loaded. The results obtained by co-loading Ps-TG3 and MK571 into GSTP1-nonexpressing cells suggest that Ps-TG3 can be a substrate for MRPs. The usefulness of Ps-TG3 was demonstrated by fluorescence imaging of several cancer cell cultures and FACS analysis of lymphoma cells. The results presented here suggest that Ps-TG3, in combination with MK571, is useful for visualizing and detecting intracellular GSTP1 activity in cancer cells that highly express GSTP1.

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity

An increase in O-GlcNAcylation of Sp1 down-regulates the gene expression of pi class glutathione S-transferase in diabetic mice

Oxidative stress is a key factor contributing to the development of diabetes complications. Glutathione S-transferases (GSTs) protect against products of oxidative stress by conjugating glutathione to electrophilic substrates, producing compounds that are generally less reactive and more soluble. The expression and activity of GSTs during diabetes have been extensively studied, but little is known about regulation mechanisms of Pi-class GST (GSTP). The aim of the present study was to evaluate how GSTP is regulated in a Streptozotocin (STZ)-induced murine diabetes model. GST activity and GSTP expression were determined in adult male mice diabetized with STZ. Specificity protein 1 (Sp1) expression and O-glycosylation, as well as the role of AP-1 members Jun and Fos in the regulation of GSTP expression, were also assessed. The results showed that GST total activity and GSTP mRNA and protein levels were decreased in the diabetic liver, and returned to normal values after insulin administration. The insulin-mimetic drug vanadate was also able to restore GST activity, but failed to recover GSTP mRNA/protein levels. In diabetic animals, O-glycosylated Sp1 levels were increased, whereas, in insulin-treated animals, glycosylation values were similar to those of controls. After vanadate administration, Sp1 expression levels and glycosylation were lower than those of controls. Our results suggest that hyperglycemia could lead to the observed increase in Sp1 O-glycosylation, which would, in turn, lead to a decrease in the expression of Sp1-dependent GSTP in the liver of diabetic mice.

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Characterization of the allergenic properties of a pi-class glutathione transferase from Ascaris suum

Characterization of the allergenic properties of a pi-class glutathione transferase from Ascaris suum

Differential responses of a pi-class glutathione S-transferase (CnGSTp) expression and antioxidant status between golden and brown noble scallops under pathogenic stress

Glutathione S-transferases (GSTs) play important roles in immunity by protecting organisms against the damage of reactive oxygen species (ROS). In this study, a pi-class GST cDNA sequence was first cloned from noble scallop Chlamys nobilis (named CnGSTp). The full length cDNA of CnGSTp was 922 bp, encoding a cytosolic protein of 202 amino acids residues, with predicted molecular masses of 23.1 kDa. Then an acute Vibrio Parahaemolyticus challenge experiment was conducted by using the Golden and Brown noble scallops with different total carotenoids content (TCC), and CnGSTp expression level, TCC and ROS level was separately determined. The results showed that ROS and CnGSTp expression levels were significantly up-regulate under Vibrio Parahaemolyticus challenge than the control group (P < 0.05). The Golden scallops showed significantly higher CnGSTp expression level and lower ROS level in hemocytes than the Brown ones (P < 0.05). Moreover, there is a significantly positive correlation between TCC and ROS in the Golden scallops. The present results revealed that CnGSTp plays important roles in immune response and carotenoids play assistant roles in antioxidant defense system under pathogenic stress in the noble scallop.

4-Bromo-1,8-naphthalimide derivatives as fluorogenic substrates for live cell imaging of glutathione S-transferase (GST) activity

Fluorogenic substrates are used to visualize the activity of cancer-associated enzymes and to interpret biological events. Certain types of glutathione S-transferase (GST), such as Pi class GST (referred to as GSTP1), are more highly expressed in a wide variety of human cancer tissues compared to their corresponding normal tissues. Pi class GST is thus a cancer cell molecular marker and potential target for overcoming resistance to chemotherapy. Here, we report that 4-bromo-1,8-naphthalimide (BrNaph) is a practical fluorogenic GST substrate. We have found that HE-BrNaph, an N-hydroxyethyl derivative, shows remarkable fluorescence enhancement upon GST-catalyzed SNAr replacement of the bromo group with a glutathionyl group. This substitution was highly selective and occurred only in the presence of GSH/GSTs; no non-enzymatic reaction was observed. We demonstrated that HE-BrNaph allows visualization of GST activity in living cells and enables to distinguish cancer cells from normal cells. Further, various N-substitutions in BrNaph retain susceptibility to enzymatic activity and isozyme selectivity, suggesting the applicability of BrNaph derivatives. Thus, BrNaph and its derivatives are GST substrates useful for fluorescence imaging and the intracellular detection of GSTP1 activity in living cells.

Short-term regulation of the hepatic activities of cytochrome P450 and glutathione S-transferase by nose-only cigarette smoke exposure in mice

The present study aimed to determine the effects of cigarette smoke on the regulation of hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes in male BALB/c mice exposed to nose-only cigarette smoke for 4 days. There were no significant increases in serum liver injury markers (alanine aminotransferase and aspartate aminotransferase) or oxidative stress (total antioxidant capacity, malondialdehyde, and glutathione disulfide/reduced glutathione) following cigarette smoke exposure, but malondialdehyde was elevated in the bronchoalveolar lavage fluid of smoke-exposed mice. Additionally, the hepatic microsomal protein levels of Cyp1a and Cyp2b, and the activities of ethoxyresorufin O-deethylase, pentoxyresorufin O-depenylase, and chlorzoxazone 6-hydrxylase, were elevated in smoke-exposed mice. Interestingly, the hepatic activities of GST toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrynic acid, but not cumene hydroperoxide were enhanced by cigarette smoke exposure, which was consistent with the increased expression levels of mu- and pi-class GSTs, but not alpha-class GSTs, observed in immunoblot analyses. These findings indicate that the short-term inhalation of cigarette smoke induces drug-metabolizing enzymes such as CYP1A, CYP2B, and mu/pi-class GSTs in the absence of hepatic injury and oxidative stress. Furthermore, smoking may alter hepatic drug metabolism, as well as the disposition and toxicity of xenobiotics, including some therapeutic drugs and cigarette smoke constituents.

Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application

Pi class glutathione S-transferase (GSTP1) is highly expressed in various cancerous cells and pre-neoplastic legions, where it is involved in apoptotic resistance or metabolism of several anti-tumour chemotherapeutics. Therefore, GSTP1 is a marker of malignant and pre-malignant cells and is a promising target for visualization and drug development. Here we demonstrate that fluorescein diacetate (FDA), a fluorescent probe used for vital staining, is a fluorescently activated by esterolytic activity of human GSTP1 (hGSTP1) selectively among various cytosolic GSTs. Fluorescence activation of FDA susceptible to GST inhibitors was observed in MCF7 cells exogenously overexpressing hGSTP1, but not in cells overexpressing hGSTA1 or hGSTM1. Inhibitor-sensitive fluorescence activation was also observed in several cancer cell lines endogenously expressing GSTP1, suggesting that GSTP1 is involved in FDA esterolysis in these cells. Among the FDA derivatives examined, FOMe-Ac, the acetyl ester of fluorescein O-methyl ether, was found to be a potential reporter for GSH-dependent GSTP1 activity as well as for carboxylesterase activity. Since GSTP1 is highly expressed in various types of cancer cells compared to their normal counterparts, improving the fluorogenic substrates to be more selective to the esterolysis activity of GSTP1 rather than carboxylesterases should lead to development of tools for detecting GSTP1-overexpressing cancer cells and investigating the biological functions of GSTP1.

Inhibition of JNK by pi class of glutathione S-transferase through PKA/CREB pathway is associated with carnosic acid protection against 6-hydroxydopamine-induced apoptosis

Pi class of glutathione S-transferase (GST) is known to suppress c-Jun N-terminal kinase (JNK)-related apoptosis through protein-protein interactions. Moreover, signaling by PKA/cAMP response element binding protein (CREB) is necessary for GSTP up-regulation. This study explored whether carnosic acid (CA) from rosemary prevents 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by inhibition of JNK through GSTP via PKA/CREB signaling. Results indicated that the GSTP protein was increased in SH-SY5Y cells treated with CA for 18 and 24 h. However, CA had no significant effect on alpha or mu class of GST. Treatment of CA increased the induction of p-PKAα, nuclear p-CREB, and CRE-DNA binding activity. These effects of CA were attenuated in cells pretreated with the PKA inhibitor H89. CA pretreatment suppressed 6-OHDA-induced apoptosis by inhibition of JNK phosphorylation, poly(ADP)-ribose polymerase cleavage, and nuclear condensation. Pretreatment with H89 and GSTP siRNA attenuated the ability of CA to reverse 6-OHDA-induced apoptosis. By use of immunoprecipitation with JNK antibody to examine the interaction of GSTP-JNK with CA, we showed that CA pretreatment increased the immunoprecipitation of GSTP after 6-OHDA treatment, which suggests that CA promoted the interaction between GSTP and JNK. Conclusion: CA prevents 6-OHDA-induced apoptosis via inhibition of JNK by GSTP through the PKA/CREB pathway.

Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical.

Dietary isothiocyanates abundant as glucosinolate precursors in many edible cruciferous vegetables are effective for prevention of cancer in chemically-induced and transgenic rodent models. Some of these agents, including phenethyl isothiocyanate (PEITC), have already advanced to clinical investigations. The primary route of isothiocyanate metabolism is its conjugation with glutathione (GSH), a reaction catalyzed by glutathione S-transferase (GST). The pi class GST of subunit type 1 (hGSTP1) is much more effective than the alpha class GST of subunit type 1 (hGSTA1) in catalyzing the conjugation. Here, we report the crystal structures of hGSTP1 and hGSTA1 each in complex with the GSH adduct of PEITC. We find that PEITC also covalently modifies the cysteine side chains of GST, which irreversibly inhibits enzymatic activity.

Identification of Differentiation-Related Proteins in Gastric Adenocarcinoma Tissues by Proteomics.

There is a significant correlation between the degree of tumor differentiation and the survival of patients with gastric cancers. In this report, we compared proteomic differences between poorly differentiated gastric adenocarcinoma tissues and well-differentiated gastric adenocarcinoma tissues in order to identify differentiation-related proteins that may be closely correlated with differentiation of gastric cancer pathogenesis. We identified 7 proteins, of which calreticulin precursor, tapasinERP57 heterodimer, pyruvate kinase isozymes M1/M2 isoform M2, class Pi glutathione S-transferase, and chain A crystal structure of human enolase 1 were upregulated in poorly differentiated gastric adenocarcinoma compared with well-differentiated gastric adenocarcinoma, while myosin-11 isoform SM2A and actin alpha cardiac were downregulated. Two of them, pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 are enzymes involved in glycolytic pathway. The upregulation of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 in poorly differentiated gastric adenocarcinoma was confirmed by Western blotting and immunohistochemistry. Furthermore, we observed 107 cases with gastric adenocarcinoma and found that the high expression of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 correlates with tumor size (P = .0001 and P = .0017, respectively), depth of invasion (P = .0024 and P = .0261, respectively), and poor prognosis of patients. In conclusion, with this proteomic analysis, pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 were identified upregulated in poorly differentiated gastric adenocarcinoma comparing with well-differentiated gastric adenocarcinoma. The expression level of pyruvate kinase isozymes M1/M2 isoform M2 and enolase 1 was significantly correlated with overall survival. Some of them would be differentiation-related cancer biomarkers and are associated with tumor metastasis, invasion, and prognosis.

A novel biomarker for marine environmental pollution of pi-class glutathione S-transferase from Mytilus coruscus

Glutathione S-transferases (GSTs) are the superfamily of phase II detoxification enzymes that play crucial roles in innate immunity. In this study, a pi-class GST homolog was identified from Mytilus coruscus (named as McGST1, KC525103). The full-length cDNA sequence of McGST1 was 621bp with a 5ʹ untranslated region (UTR) of 70bp and a 3ʹ-UTR of 201bp. The deduced amino acid sequence was 206 residues in length with theoretical pI/MW of 5.60/23.72kDa, containing the conserved G-site and diversiform H-site. BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of pi class GST family. The prediction of secondary structure displayed a preserved N-terminal and a C-terminal comprised with α-helixes. Quantitative real time RT-PCR showed that constitutive expression of McGST1 was occurred, with increasing order in mantle, muscle, gill, hemocyte, gonad and hepatopancreas. The stimulation of bacterial infection, heavy metals and 180CST could up-regulate McGST1 mRNA expression in hepatopancreas with time-dependent manners. The maximum expression appeared at 6h after pathogenic bacteria injected, with 10-fold in Vibrio alginolyticus and 16-fold in Vibrio harveyi higher than that of the control. The highest point of McGST1 mRNA appeared at different time for exposure to copper (10-fold at day 15), cadmium (9-fold at day10) and 180 CST (10-fold at day 15). These results suggested that McGST1 played a significant role in antioxidation and might potentially be used as indicators and biomarkers for detection of marine environmental pollution.

Proteomic analysis of kidney in rats chronically exposed to monosodium glutamate.

BackgroundChronic monosodium glutamate (MSG) intake causes kidney dysfunction and renal oxidative stress in the animal model. To gain insight into the renal changes induced by MSG, proteomic analysis of the kidneys was performed.MethodsSix week old male Wistar rats were given drinking water with or without MSG (2 mg/g body weight, n = 10 per group) for 9 months. Kidneys were removed, frozen, and stored at –75°C. After protein extraction, 2-D gel electrophoresis was performed and renal proteome profiles were examined with Colloidal Coomassie Brilliant Blue staining. Statistically significant protein spots (ANOVA, p<0.05) with 1.2-fold difference were excised and analyzed by LC-MS. Proteomic data were confirmed by immunohistochemistry and Western blot analyses.ResultsThe differential image analysis showed 157 changed spots, of which 71 spots were higher and 86 spots were lower in the MSG-treated group compared with those in the control group. Eight statistically significant and differentially expressed proteins were identified: glutathione S-transferase class-pi, heat shock cognate 71 kDa, phosphoserine phosphatase, phosphoglycerate kinase, cytosolic glycerol-3-phosphate dehydrogenase, 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, α-ketoglutarate dehydrogenase and succinyl-CoA ligase.ConclusionThe identified proteins are mainly related to oxidative stress and metabolism. They provide a valuable clue to explore the mechanism of renal handling and toxicity on chronic MSG intake.