Structural and functional consequences of haloenol lactone inactivation of murine and human glutathione S-transferase

Mass spectrometric analysis of proteolysis products of haloenol lactone-modified glutathione S-transferase isozyme mGSTP1 indicates that the haloenol lactone 3-cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone is covalently attached to the protein at Cys-47. Comparisons of the extent of adduct formation with losses in enzymatic activity indicate that mGSTP1 exhibits greatest reactivity toward the haloenol lactone, followed by mGSTM1 and mGSTA3. Activities of mGSTP1 and mGSTM1 decrease in inverse proportion to haloenol lactone concentration, whereas modification had no apparent effect on catalytic activity of mGSTA3. Decreases in activity agree with the extent of protein modification observed in ESI mass spectra for mGSTP1 and mGSTM1 but not for mGSTA3. Kinetic studies employing recombinant human proteins with replacement of cysteine by serine at Cys-47 and Cys-101 indicate that rapid inactivation (t1/2 = 2 min) occurs only when residue 47 is cysteine. Mass spectra of C47S-hGSTP1 incubated with haloenol lactone demonstrate covalent attachment of a haloenol lactone-glutathione conjugate and suggest that an ester forms between the lactone and Ser-47. Therefore, we propose that initial opening of the lactone ring is promoted by Cys-47 through thioester formation between the lactone carbonyl and the Cys-47 sulfhydryl. Enol-keto tautomerization and enzyme-mediated hydrolytic cleavage of the thioester produces a reactive alpha-bromoketone which reacts a second time with Cys-47 and inactivates the enzyme. These results suggest that Pi class GSTs have thioesterase activity and that haloenol lactone inactivation occurs through an enzyme-mediated process.     

Selective expression of a glutathione S-transferase subclass during spermatogenesis

Levels of the hGSTM3 glutathione S-transferase (GST) subunit in testis of the human fetus and infant were found to be only a small fraction of those in adults. To understand these observations and to determine whether hGSTM3 subunit expression is developmentally and/or hormonally regulated, an experimental model based on the rat testis homologue (subunit rGSTM5) was used. For prepubertal rats, testicular rGSTM5 subunit levels were very low, but a sharp increase was observed between weeks 6 and 7 of development, when testicular growth includes increased numbers of germ cells associated with spermatogenesis. In adult hypophysectomized rats, the rGSTM5 subunit content of testis decreased progressively over 5 weeks, at which time the subunit was barely detectable. In contrast, the other GST subunit types did not vary significantly during development or after hypophysectomy. These results suggest that rGSTM5 subunits in rat testis could originate from spermatogenic cells. Accordingly, GSTs were purified from human sperm, and it was shown that the hGSTM3 subunit was, by a large measure, the predominant form. These data are consistent with the notion that the differential expression of hGSTM3 during human testicular development can also be explained on the basis of its preferential location in germs cells.     

Synthesis and glutathione S-transferase structure-affinity relationships of nonpeptide and peptidase-stable glutathione analogues

A series of nonpeptidic glutathione analogues where the peptide bonds were replaced by simple carbon-carbon bonds or isosteric E double bonds were prepared. The optimal length for the two alkyl chains on either side of the mercaptomethyl group was evaluated using structure-affinity relationships. Affinities of the analogues 14a-f, 23, and 25 were evaluated for a recombinant GST enzyme using a new affinity chromatography method previously developed in our laboratory. Analysis of these analogues gives an additional understanding for GST affinity requirements: (a) the carbon skeleton must conserve that of glutathione since analogue 14a showed the best affinity (IC50 = 5.2 microM); (b) the GST G site is not able to accommodate a chain length elongation of one methylene group (no affinity for analogues 14c,f); (c) a one-methylene group chain length reduction is tolerated, much more for the "Glu side" (14d, IC50 = 10.1 microM) than for the "Gly side" (14b, IC50 = 1800 microM); (d) the mercaptomethyl group must remain at position 5 as shown from the null affinity of the 6-mercaptomethyl analogue 14e; (e) the additional peptide isosteric E double bond (25) or hydroxyl derivative (23) in 14e did not help to retrieve affinity. This work reveals useful information for the design of new selective nonpeptidic and peptidase-stable glutathione analogues.     

Structural studies of a human pi class glutathione S-transferase. Photoaffinity labeling of the active site and target size analysis

The present study analysed gubernaculum development in mice that had been induced, through transgenesis, to express human anti-Müllerian hormone (h-AMH) throughout prenatal life. Growth and differentiation of the gubernacular primordia were assessed through the analysis of serial, transverse or sagittal, histological sections of the lower abdomen. Transgenic males and females expressed biologically active amounts of h-AMH as measured by sensitive and specific ELISA and evidenced through the regression, in females, of Müllerian ducts after day 13 of prenatal life. Gubernacular primordia became distinguishable at the same age in control and transgenic male and female fetuses on day 12 after coitus. In both groups gubernacular cords (inguinal folds of the genital mesenteries) increased in length more in females than in males while gubernacular cones showed larger growth in males. h-AMH thus appeared not to affect the sexually dimorphic pattern of growth and development of these structures. Growth and differentiation of the gubernacular primordia was further examined in 18-day-old control and h-AMH transgenic fetuses that had been exposed to testosterone propionate injected into their mothers on days 12 and 14 of pregnancy. Testosterone treatment affected, to a minor extent, the growth of the female gubernacular cords: these were reduced in length (but had a larger surface area) compared with controls. The gubernacular cones were slightly increased in length but male-like differentiation of the tissues of the cones into a muscular and mesenchymal component was not noticed to any extent. The observations thus add experimental support to the contention that AMH, even in combination with testosterone, is not effective in establishing the male pattern of gubernacular primordia development.     

Suramin is an active site-directed, reversible, and tight-binding inhibitor of protein-tyrosine phosphatases

The effect of suramin, a well known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers, on protein-tyrosine phosphatases (PTPases) has been examined. Suramin is a reversible and competitive PTPase inhibitor with Kis values in the low microM range, whereas the Kis for the dual specificity phosphatase VHR is at least 10-fold higher. Although suramin can also inhibit the activity of the potato acid phosphatase at a slightly higher concentration, it is 2-3 orders of magnitude less effective against the protein Ser/Thr phosphatase 1alpha and the bovine intestinal alkaline phosphatase. Suramin binds to the active site of PTPases with a binding stoichiometry of 1:1. Furthermore, when suramin is bound to the active site of PTPases, its fluorescence is enhanced approximately by 10-fold. This property has allowed the determination of the binding affinity of suramin for PTPases and several catalytically impaired mutant PTPases by fluorescence titration techniques. Thus, the active site Cys to Ser mutants bind suramin with similar affinity as the wild type, while the active site Arg to Ala mutant exhibits a 20-fold reduced affinity toward suramin. Interestingly, the general acid deficient Asp to Ala mutant PTPases display an enhanced affinity toward suramin, which is in accord with their use as improved "substrate-trapping" agents. That suramin is a high affinity PTPase inhibitor is consistent with the observation that suramin treatment of cancer cell lines leads to an increase in tyrosine phosphorylation of several cellular proteins. Given the pleiotropic effects of suramin on many enzyme systems and growth factor-receptor interactions, the exact in vivo actions of suramin require further detailed structure-activity investigation of suramin and its structural analogs.     

trans-2-phenylcyclopropylamine is a substrate for and inactivator of horseradish peroxidase

Horseradish peroxidase (HRP) is well known for mediating the electron-transfer oxidation of electron-rich aromatic 'donors' such as phenols and anilines, but has not been described to oxidize aliphatic amines. We here confirm the inability of HRP to oxidize typical aliphatic amines, even those which would exist significantly as free bases at the operative pH. In contrast, trans-2-phenylcyclopropylamine (2-PCPA) is both a substrate (turnover product is cinnamaldehyde) and a time-dependent inactivator of HRP. These activities of 2-PCPA are consistent with either a concerted or rapid sequential one-electron-oxidation/ring-opening to give an intermediate capable of covalent binding to the enzyme. 2-PCPA is the first known example of a simple aliphatic amine which serves as a substrate for HRP under turnover conditions.     

Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis

Prolyl aminopeptidase (PAP) has been classified as a sulfhydryl enzyme on the basis of its high sensitivity to p-chloromercuribenzoate and heavy metals. Recently, however, the possibility of PAP being instead a serine enzyme has been raised as a result of two observations--the conservation of some residues among the PAPs hitherto sequenced, and a similarity to some serine hydrolases. This is the first report describing an attempt to identify the active residue by site-directed mutagenesis. The pap genes from Bacillus coagulans and Aeromonas sobria, were used for the study. The changes made were Cys62Ser and Ser101Ala for the first enzyme, and Thr92Ala and Ser146Ala for the second. For both enzymes, only the changes made on the serine residues resulted in their complete inactivation, indicating that PAP is a serine peptidase.     

Induction of murine hepatic glutathione S-transferase by dietary dehydroepiandrosterone

The naturally occurring steroid dehydroepiandrosterone (DHEA), when administered as a supplement to the diet of mice and rats, produces alterations in the relative concentrations of specific liver proteins; among these, a protein of Mr approximately 28 K is markedly induced by DHEA action. In the present study we identified the murine hepatic approximately 28 kDa protein as glutathione S-transferase subtype GT-8.7. Glutathione S-transferases belong to a gene superfamily that encode closely related proteins which are induced in liver and other tissues by various chemicals, including carcinogens and chemoprotective agents such as dietary antioxidants. Based on the above finding, we evaluated glutathione S-transferase activity in cytosols and microsomes prepared from liver tissue of mice fed either a control diet or a DHEA-containing diet (0.45%, by weight). The specific activity of hepatic cytosolic glutathione S-transferase in mice treated with DHEA up to 7 days was either unchanged or slightly decreased when compared to controls; however, treatment for 14 days or longer resulted in significant increases in activity. The specific activity of microsomal glutathione S-transferase also was increased by long-term DHEA treatment; however, its activity was approximately one-tenth of that in corresponding cytosols.     

Induction of glutathione S-transferase activity by curcumin in mice

Curcumin, a natural constituent of Curcuma longa (turmeric, CAS 458-37-7), has been studied for its induction of glutathione S-transferase activity in mice. At a dose of 250 mg/kg orally for 15 days, the enzyme activity in liver was increased by 1.8 fold. Its effect on other tissues like stomach, small intestine, lungs, kidney was not significant. Curcumin also depleted sulfhydryl levels in tissues, especially in stomach where 45% depletion was observed.     

Overexpression of glutathione S-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress

Transgenic tobacco seedlings that overexpress a cDNA encoding an enzyme with both glutathione S-transferase (GST) and glutathione peroxidase (GPX) activity had GST- and GPX-specific activities approximately twofold higher than wild-type seedlings. These GST/GPX overexpressing seedlings grew significantly faster than control seedlings when exposed to chilling or salt stress. During chilling stress, levels of oxidized glutathione (GSSG) were significantly higher in transgenic seedlings than in wild-types. Growth of wild-type seedlings was accelerated by treatment with GSSG, while treatment with reduced glutathione or other sulfhydryl-reducing agents inhibited growth. Therefore, overexpression of GST/GPX can stimulate seedling growth under chilling and salt stress, and this effect could be caused by oxidation of the glutathione pool.     

Tumor efficacy and bone marrow-sparing properties of TER286, a cytotoxin activated by glutathione S-transferase

TER286 is a latent drug activated by human glutathione S-transferase (GST) isoforms P1-1 and A1-1 to produce a nitrogen mustard alkylating agent. M7609 human colon carcinoma, selected for resistance to doxorubicin, and MCF-7 human breast carcinoma, selected for resistance to cyclophosphamide, both showed increased sensitivity to TER286 over their parental lines in parallel with increased expression of GST P1-1. In primary human tumor clonogenic assays, the spectrum of cytotoxic activity observed for TER286 was both broad and unusual when compared to a variety of current drugs. In murine xenografts of M7609 engineered to have high, medium, or low GST P1-1, responses to TER286 were positively correlated with the level of P1-1. Cytotoxicity was also observed in several other cell culture and xenograft models. In xenografts of the MX-1 human breast carcinoma, tumor growth inhibition or regression was observed in nearly all of the animals treated with an aggressive regimen of five daily doses. This schedule resulted in a 24-h posttreatment decline in bone marrow progenitors to 60% of control and was no worse than for a single dose of TER286. These studies have motivated election of TER286 as a clinical candidate.     

Catalytic mechanism and role of hydroxyl residues in the active site of theta class glutathione S-transferases. Investigation of Ser-9 and Tyr-113 in a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina

Spectroscopic and kinetic studies have been performed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyperbolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that product release is rate-limiting. Spectroscopic and kinetic data demonstrate that Lucilia GST is able to lower the pKa of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pKa of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH activation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on alpha-helix 4, and its hydroxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of alpha-helix 4 possibly modulates different aspects of the catalytic mechanism of the Lucilia GST.     

Crystal structure of a murine alpha-class glutathione S-transferase involved in cellular defense against oxidative stress

STATEMENT OF PROBLEM: The electric alloy brazed joints of removable partial denture alloys have failed frequently after routine usage. PURPOSE: A technique providing higher joint strengths was investigated. This investigation compared the tensile strengths of electric-brazed and laser-welded joints for a cobalt-chromium removable partial denture alloy. MATERIAL AND METHODS: Twenty-four cobalt-chromium standard tensile testing rods were prepared and divided into three groups of eight. All specimens in the control group (group 1) were left in the as-cast condition. Groups 2 and 3 were the test specimens, which were sectioned at the center of the rod. Eight specimens were joined by using electric brazing, and the remaining specimens were joined by using laser welding. After joining, each joint was ground to a uniform diameter, then tested to tensile failure on an Instron universal testing machine. Failure loads were recorded and fracture stress calculated. Statistical analysis was applied. RESULTS: The student-Newman-Keuls test showed a highly significant difference between the joint strengths of the as-cast control specimens, the electric-brazed and laser-welded joints. CONCLUSIONS: The tensile strengths of the as-cast joints were higher than those for the laser-welded joints, and both were higher than the electric-brazed joint strengths.     

Role of cellular glutathione and glutathione S-transferase in the expression of alkylating agent cytotoxicity in human breast cancer cells

Glutathione (GSH) and glutathione S-transferases (GSTs) play an important role in the protection of cells against toxic effects of many electrophilic drugs and chemicals. Modulation of cellular GSH and/or GST activity levels provides a potentially useful approach to sensitizing tumor cells to electrophilic anti-cancer drugs. In this study, we describe the interactions of four representative alkylating agents (AAs), melphalan, 4-hydroperoxy-cyclophosphamide (4HC), an an activated form of cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and cisplatin, with GSH and GST in the human breast cancer cell line MCF-7. Depletion of cellular GSH pools by approximately 80% by treatment of the cells with the GSH synthesis inhibitor buthionine sulfoximine (BSO) sensitized the tumor cells to each AA to a different extent, with dose-modifying factors of 2.39, 2.21, 1.64, and 1.27 observed for melphalan, 4HC, cisplatin, and BCNU, respectively. Treatment of the cells with the GST inhibitor ethacrynic acid (EA) failed to show any significant effects on the cytotoxicity of these AAs. However, EA did potentiate the cytotoxicity of melphalan when given in combination with BSO, an effect that may be due to a more complete depletion of cellular GSH levels by the combined modulator treatment. Following a 1-hr exposure to cytotoxic-equivalent concentrations of these AAs, GSH levels decreased substantially in the case of 4HC and BCNU, but increased by 30-50% in the case of cisplatin and melphalan. BSO pretreatment largely blocked this effect of cisplatin and melphalan on cellular GSH, while it further enhanced the GSH-depleting activity of both 4HC and BCNU. On the basis of these results, it is concluded that (a) GSH affects the cytotoxicity of different AAs to different extents, (b) basal GST expression in MCF-7 cells does not play a major role in AA metabolism, (c) EA can potentiate the enhancing effect of BSO on melphalan cytotoxicity in MCF-7 cells, and (d) depletion of cellular GSH by pretreatment with BCNU or cyclophosphamide may correspond to a useful strategy for enhancing the anti-tumor activity of other AAs given in a sequential combination.     

Alpha,beta-unsaturated carbonyl compounds: inhibition of rat liver glutathione S-transferase isozymes and chemical reaction with reduced glutathione

Monoclonal antibodies have been obtained by fusing mouse myeloma cells with spleen cells of mice immunized with crude thyroid membranes. Among the antibodies reactive with different thyroid antigenic components, three were found to specifically react with TSH receptor molecules. These antibodies displayed characteristic staining patterns on frozen sections of thyroid tissue from patients with various thyroid diseases upon identification of antibody binding by indirect peroxidase staining. No specific reactivity was detected with tissue from other human organs, such as pancreas, liver, fat, and muscle. The results demonstrate that the immunoperoxidase technique and the specificity of the monoclonal antibodies produced permitted the identification of cellular constituents that might be important antigens in autoimmune thyroid disease.     

Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.     

Methionine and cysteine affect glutathione level, glutathione-related enzyme activities and the expression of glutathione S-transferase isozymes in rat hepatocytes

Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and gamma-glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P < 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.     

Structure and organization of the human alpha class glutathione S-transferase genes and related pseudogenes

We have isolated and characterized genomic DNA encoding several human Alpha class glutathione S-transferase genes and pseudogenes. All the genes are composed of seven exons with boundaries identical to those of the Alpha class genes in rats. The GSTA1 gene is approximately 12 kb in length and is closely flanked by other Alpha class gene sequences. The complete sequence of the 1.7-kb intergenic region between exon 7 of an upstream pseudogene and exon 1 of the GSTA1 gene has been determined. An additional gene that encodes an uncharacterized Alpha class glutathione S-transferase has been identified. The protein derived from this gene would have 19 amino acid substitutions compared with the GSTA1 isoenzyme. Several pseudogenes with single-base and/or complete exon deletions have been identified, but no reverse-transcribed pseudogenes have been detected. The occurrence of multiple genes and pseudogenes on a single fragment of cloned genomic DNA and the prior identification of a single chromosomal region (6p12) of hybridization (Board and Webb, 1987, Proc. Natl. Acad. Sci. USA 84:2377-2381) suggest that all the Alpha class genes are members of a closely linked gene family that has evolved by duplication and gene conversion events.