Equilibrium unfolding of class pi glutathione S-transferase

The equilibrium unfolding transition of class pi glutathione S-transferase, a homodimeric protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded dimer and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, delta Gu (H2O), of the native dimer was estimated to be about 25.3 +/- 2 kcal/mol at 20 degrees C and pH6.5.     

Triiodothyronine downregulates the periportal expression of alpha class glutathione S-transferase in rat liver

Most drug-metabolizing phase I and phase II enzymes, including the glutathione S-transferases (GST), exhibit a zonated expression in the liver, with lower expression in the upstream, periportal region. To elucidate the involvement of pituitary-dependent hormones in this zonation, the effect of hypophysectomy and 3,3',5-triiodo-L-thyronine (T3) on the distribution of GST was studied in rats. Hypophysectomy increased total GST activity both in the periportal and perivenous liver region. Subsequent T3 treatment counteracted this effect in the perivenous zone. However, analysis for either mu class M1/M2-specific (1,2-dichloro-4-nitrobenzene) or alpha class A1/A2-specific (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) GST activity revealed that T3 treatment did not significantly affect the perivenous activity of these GST classes. In contrast, T3 was found to significantly counteract the increase of alpha class GST activity caused by hypophysectomy in the periportal zone. To establish whether this effect was T3-specific, hepatocytes were isolated from either the periportal and perivenous zone by digitonin/collagenase perfusion and cultured either as pyruvate-supplemented monolayer or as co-culture with rat liver epithelial cells. Only in the latter it was found that T3 suppressed the A1/A2-specific GST activity and alpha class proteins predominantly in periportal cells. The data demonstrate that T3 is an important factor responsible for the low expression of alpha GST in the periportal region. T3 may be involved in the periportal downregulation of other phase I and II enzymes as well.     

An alpha class glutathione S-transferase from pike liver

Glutathione S-transferase (GST) was isolated from the Esox lucius liver and purified to the homogenous state by SDS-PAGE and isoelectrofocusing. It was found to be a homodimer with a subunit molecular weight of 25235.36 Da (HPLC-MS/MS data) and pI of about 6.4. Its substrate specificity, thermal stability, some kinetic characteristics, and optimum pH were studied. The enzyme was identified as Alpha class GST.     

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration

Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.     

Differential expression of alpha and pi isoenzymes of glutathione S-transferase in developing human kidney

Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies to determine the developmental expression of these isoforms in human kidney. Before 35 weeks of gestation, both isoenzymes were expressed by the collecting tubules and developing nephrons. After this time, expression of the alpha set was restricted to the proximal tubule and that of the pi set to the distal and collecting tubules and the loop of Henle.     

The three-dimensional structure of class pi glutathione S-transferase in complex with glutathione sulfonate at 2.3 A resolution

The three-dimensional structure of class pi glutathione S-transferase from pig lung, a homodimeric enzyme, has been solved by multiple isomorphous replacement at 3 A resolution and preliminarily refined at 2.3 A resolution (R = 0.24). Each subunit (207 residues) is folded into two domains of different structure. Domain I (residues 1-74) consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side, facing the solvent, by a bent, irregular helix structure. The topological pattern resembles the bacteriophage T4 thioredoxin fold, in spite of their dissimilar sequences. Domain II (residues 81-207) contains five alpha-helices. The dimeric molecule is globular with dimensions of about 55 A x 52 A x 45 A. Between the subunits and along the local diad, is a large cavity which could possibly be involved in the transport of nonsubstrate ligands. The binding site of the competitive inhibitor, glutathione sulfonate, is located on domain I, and is part of a cleft formed between intrasubunit domains. Glutathione sulfonate is bound in an extended conformation through multiple interactions. Only three contact residues, namely Tyr7, Gln62 and Asp96 are conserved within the family of cytosolic glutathione S-transferases. The exact location of the binding site(s) of the electrophilic substrate is not clear. Catalytic models are discussed on the basis of the molecular structure.     

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.     

Nucleotide sequence of a class mu glutathione S-transferase from chicken liver

A clone coding for glutathione S-transferase (GST) CL2 was isolated from a chicken liver cDNA library. This clone (819 bp) encodes a polypeptide comprising 219 amino acids with a molecular weight of 25,717, excluding the initiator methionine. The primary amino acid sequence of the enzyme has 47% identical sequence with other class mu GSTs.     

HDAC1 inhibition by maspin abrogates epigenetic silencing of glutathione S-transferase pi in prostate carcinoma cells

Both maspin and glutathione S-transferase pi (GSTp) are implicated as tumor suppressors and downregulated in human prostate cancer. It is well established that GSTp downregulation is through DNA methylation-based silencing. We report here that maspin expression in prostate cancer cell line DU145 reversed GSTp DNA methylation, as measured by methylation- specific PCR, MethyLight assay, and bisulfite sequencing. The effect of maspin on GSTp expression was similar to that of the combination of a synthetic histone deacetylase (HDAC) inhibitor and DNA methylation inhibitor 5-aza-2'-deoxycytidine. Maspin expression also led to an increased level of acetylated histone 3, decreased level of methyl transferase, and methyl-CpG-binding domain proteins at the site of demethylated GSTp promoter DNA. Earlier, we have shown that maspin inhibits HDAC1. In PC3 cells, where both maspin and GSTp are expressed at a reduced level, maspin knockdown led to a significant reduction in GSTp expression, whereas dual knockdown of maspin and HDAC1 barely increased the level of GSTp expression. Thus, HDAC1 may play an essential role in cellular response to maspin-mediated GSTp desilencing. Maspin has been shown to increase tumor cell sensitivity to drug-induced apoptosis. Interestingly, GSTp reexpression in the absence of maspin expression perturbation blocked the phosphorylation of histone 2A.X, the induction of hypoxia-induced factor 1α (HIF-1α), and cell death of LNCaP cells under oxidative stress. Because DNA hypermethylation-based silencing may couple with and depend on histone deacetylation, our study suggests that endogenous HDAC inhibition by maspin may prevent pathologic gene silencing in prostate tumor progression.     

Tissue-specific expression and subcellular distribution of murine glutathione S-transferase class kappa.

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1-1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.     

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.     

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M_w of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M_w of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K_m value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer.     

Primary and secondary structural analyses of glutathione S-transferase pi from human placenta

The primary structure of glutathione S-transferase (GST) pi from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626-5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST pi genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST pi (residues 155-181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of pi, one of mu, two of alpha, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.     

Novel class of bivalent glutathione S-transferase inhibitors

Exploiting the principle of bivalent binding, we have designed symmetrical, bifunctional inhibitors to simultaneously occupy both active sites of cytosolic glutathione S-transferase, with enhanced specificity for the P1-1 isoform. We have prepared two series of compounds that differ in their binding domains-the first is a series of bis-glutathione conjugates, and the second is a series of compounds each possessing two equivalents of Uniblue A, an analogue of Cibacron Blue. For each series, a monofunctional reference compound was also prepared to determine the relative advantage of the bivalent inhibitors. Within each series, the most potent inhibitors exhibited IC(50) values 2 orders of magnitude lower than the relevant reference compounds. Moreover, within the bis-glutathionyl series, a 10-fold increase in selectivity was achieved for GST P1-1 over the A1-1 isoform. Isothermal titration calorimetry with a representative bis-glutathione conjugate and a monofunctional reference compound indicates that the bivalent inhibitor exhibits the expected increase in intrinsic affinity and decrease in stoichiometry relative to the monofunctional compound, supporting the overall design strategy.     

P-glycoprotein, glutathione and glutathione S-transferase increase in a colon carcinoma

The acquisition of resistance to anticancer agents used in chemotherapy is the main cause of treatment failure in malignant disorders, provoking tumours to become resistant during treatment, although they initially respond to it. The main multidrug resistance (MDR) mechanism in tumour cells is the expression of P-gly-coprotein (P-gly), that acts as an ATP-dependent active efflux pump of chemotherapeutic agents. Furthermore, an increased detoxification of compounds mediated by high levels of glutathione (GSH) and glutathione S-transferase (GST), has been found in resistant cells. We developed a study aiming to evaluate the evolution of the main drug resistance markers in tumour cells: P-gly, GSH and GST, during the acquisition of resistance to colchicine, for the purpose of studying the adaptation process and its contribution to the MDR phenomenon. A human colon adenocarcinoma cell line was exposed to colchicine during 82 days, being P-gly, GSH levels and GST activity evaluated by flow cytometry, spectrofluorimetry and spectrophotometry, during exposure time. P-gly and GSH levels increased gradually during the exposure to colchicine, reaching 2.35 and 3.21 fold each. On day 82, GST activity increased 1.84 fold at the end of the exposure period. Moreover, an increment in drug cross-resistance was obtained that ranges from 2.62 to 5.22 fold for colchicine, vinblastine, vincristine and mitomycin C. The increments obtained in P-gly, GSH and GST could probably contribute to the MDR phenomenon in this human colon adenocarcinoma cell line.     

Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.