Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin

In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon 1 h incubation with 100 microM quercetin or 2 h incubation with 25 microM quercetin, whereas 1 and 10 microM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LC-MS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LC-MS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47.     

Detection and biochemical characterisation of a novel polymorphism in the human GSTP1 gene

The glutathione transferases (GSTs) mediate the detoxification of a broad spectrum of electrophilic chemicals. We report here the identification and characterisation of a novel naturally occurring transition that changes codon 169 from GGC (Gly) to GAC (Asp) in the human Pi class GST, GSTP1. Expression of the variant in human HepG2 cells led to a small increase in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation compared to the wild-type protein. Asp(169) GSTP1-1 expressed at high levels in Escherichia coli displayed a small but significant increase in specific activity towards CDNB compared to Gly(169) GSTP1-1. The catalytic efficiency with CDNB was higher for Asp(169) GSTP1-1 compared to the wild-type enzyme, although the kinetic constants of the mutant and the wild-type enzyme towards glutathione were not different. Modelling indicated that the mutation does not appear to change protein conformation. The distribution of the genotypes in a normal healthy population (217 individuals) was 94.3% for the Gly/Gly genotype and 5.7% for the Gly/Asp genotype; no Asp/Asp genotypes were detected in this population. The frequency of the Asp(169) allele in the only oxidative stress-linked pathology that we have studied to date, i.e. alcoholic liver disease, was not significantly different from healthy controls. In conclusion, we have detected and characterised a novel SNP in GSTP1 that may play a role in modulating the activity of GSTP1-1.     

Detection and biochemical characterisation of a novel polymorphism in the human GSTP1 gene

The glutathione transferases (GSTs) mediate the detoxification of a broad spectrum of electrophilic chemicals. We report here the identification and characterisation of a novel naturally occurring transition that changes codon 169 from GGC (Gly) to GAC (Asp) in the human Pi class GST, GSTP1. Expression of the variant in human HepG2 cells led to a small increase in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation compared to the wild-type protein. Asp¹⁶⁹ GSTP1-1 expressed at high levels in Escherichia coli displayed a small but significant increase in specific activity towards CDNB compared to Gly¹⁶⁹ GSTP1-1. The catalytic efficiency with CDNB was higher for Asp¹⁶⁹ GSTP1-1 compared to the wild-type enzyme, although the kinetic constants of the mutant and the wild-type enzyme towards glutathione were not different. Modelling indicated that the mutation does not appear to change protein conformation. The distribution of the genotypes in a normal healthy population (217 individuals) was 94.3% for the Gly/Gly genotype and 5.7% for the Gly/Asp genotype; no Asp/Asp genotypes were detected in this population. The frequency of the Asp¹⁶⁹ allele in the only oxidative stress-linked pathology that we have studied to date, i.e. alcoholic liver disease, was not significantly different from healthy controls. In conclusion, we have detected and characterised a novel SNP in GSTP1 that may play a role in modulating the activity of GSTP1-1.     

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

Glutathione transferase P1-1 is over expressed in some cancer cells and contributes to detoxification of anticancer drugs, leading to drug-resistant tumors. The inhibition of human recombinant GSTP1-1 by natural plant products was investigated using 10 compounds isolated from plants indigenous to Southern and Central Africa. Monochlorobimane and 1-chloro-2,4-dinitrobenzene were used to determine GST activity. Each test compound was screened at 33 and 100 μM. Isofuranonapthoquinone (1) (from Bulbine frutescens) showed 68% inhibition at 33 μM, and sesquiterpene lactone (2) (from Dicoma anomala) showed 75% inhibition at 33 μM. The IC(50) value of 1 was 6.8 μM. The mode of inhibition was mixed, partial (G site) and noncompetitive (H site) with K(i) values of 8.8 and 0.21 μM, respectively. Sesquiterpene 2 did not inhibit the CDNB reaction. Therefore, isofuranonapthoquinone 1 needs further investigations in vivo because of its potent inhibition of GSTP1-1 in vitro.     

The action of the glutathione transferase substrate, 1-chloro-2,4-dinitrobenzene on synaptosomal glutathione content and the release of hydrogen peroxide

We studied the action of the glutathione transferase substrate, 1-chloro-2,4-dinitrobenzene (CDNB) on the synaptosomal production of H2O2. We found that CDNB (30-40 microM) readily depletes the cytosolic glutathione but is almost without effect on the mitochondrial fraction. The depletion of the cytosolic glutathione induced by CDNB affords the detection in the extracellular space of H2O2 produced intrasynaptosomally upon increasing the cytosolic Ca2+ concentration that is otherwise destroyed by glutathione peroxidase. Higher concentrations of CDNB induce a H2O2 production which is not related to the glutathione content. This H2O2 is of mitochondrial origin and requires that NAD be reduced. The primary product of the mitochondrial CD-NB-dependent oxygen reduction is at least in part the superoxide anion.     

Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism.

Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.     

Saccharomyces cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol transferases

The Saccharomyces cerevisiae genome encodes three proteins that display similarities with human GSTOs (Omega class glutathione S-transferases) hGSTO1-1 and hGSTO2-2. The three yeast proteins have been named Gto1, Gto2 and Gto3, and their purified recombinant forms are active as thiol transferases (glutaredoxins) against HED (beta-hydroxyethyl disulphide), as dehydroascorbate reductases and as dimethylarsinic acid reductases, while they are not active against the standard GST substrate CDNB (1-chloro-2,4-dinitrobenzene). Their glutaredoxin activity is also detectable in yeast cell extracts. The enzyme activity characteristics of the Gto proteins contrast with those of another yeast GST, Gtt1. The latter is active against CDNB and also displays glutathione peroxidase activity against organic hydroperoxides such as cumene hydroperoxide, but is not active as a thiol transferase. Analysis of point mutants derived from wild-type Gto2 indicates that, among the three cysteine residues of the molecule, only the residue at position 46 is required for the glutaredoxin activity. This indicates that the thiol transferase acts through a monothiol mechanism. Replacing the active site of the yeast monothiol glutaredoxin Grx5 with the proposed Gto2 active site containing Cys46 allows Grx5 to retain some activity against HED. Therefore the residues adjacent to the respective active cysteine residues in Gto2 and Grx5 are important determinants for the thiol transferase activity against small disulphide-containing molecules.     

Inhibition of 20 S and 26 S proteasome activity by lithium chloride: impact on the differentiation of leukemia cells by all-trans retinoic acid

Lithium affects several enzymatic activities, however, the molecular mechanisms of lithium actions are not fully understood. We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D(+) cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor alpha protein pools. Here, we demonstrate the effects of LiCl on proteasome-dependent degradation of retinoic acid receptor alpha proteins. LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor alpha and markedly reduced chymotryptic-like activity of WEHI-3B D(+) 20 S and 26 S proteasome enzymes. Neither KCl nor all-trans-retinoic acid affected enzyme activity, whereas NaCl produced a modest reduction at relatively high concentrations. In addition, LiCl inhibited 20 S proteasome chymotryptic-like activity from rabbits but had no effect on tryptic-like activity of the 26 S proteasome. This effect has significant consequences in stabilizing the retinoic acid receptor alpha protein levels that are necessary to promote continued differentiation of leukemia cells in response to all-trans-retinoic acid. In support of this concept, combination of proteasome inhibitors beta-clastolactacystin or benzyloxycarbonyl-Leu-Leu-Phe with all-trans-retinoic acid increased differentiation of WEHI-3B D(+) cells in a manner that was analogous to the combination of LiCl and all-trans-retinoic acid.     

Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein

By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha 9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha 9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters K(cat) and K(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha 9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on K(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus

Analysis of the genome of the human pathogen, Aspergillus fumigatus, revealed the presence of several putative glutathione transferase (GST) open reading frames. Three A. fumigatus GST genes, termed gstA, B, and C, were cloned and recombinant proteins expressed in Escherichia coli. Functional analysis of recombinant gstA-C confirms that the enzymes exhibit GST activity and glutathione peroxidase activity. RT-PCR confirmed low basal expression of gstA and gstC which was markedly up-regulated (at least 4x-10x) in the presence of either H2O2 or 1-chloro-2,4-dinitrobenzene (CDNB). GstB expression was only observed in the presence of CDNB. These results demonstrate for the first time the existence of three functional GSTs in A. fumigatus and strongly suggest a role for these enzymes in the response of the organism to both oxidative stress and xenobiotic presence.     

The synthesis of alpha,beta-unsaturated carbonyl derivatives with the ability to inhibit both glutathione S-transferase P1-1 activity and the proliferation of leukemia cells

Ethacrynic acid (EA), an alpha,beta-unsaturated carbonyl compound, is a glutathione S-transferase P1-1 (GSTP1-1) inhibitor. Twenty-one novel EA derivatives have been synthesized. The effects of these compounds on GSTP1-1 activity and on the proliferation of human leukemia HL-60 cells have been determined. Compounds with a halogen substitution at the 3'-position of the aromatic ring have greater inhibitory effects on GSTP1-1 activity than those of compounds with a methyl substitution there. Compounds with substitutions at both the 2'- and 3'-positions of the aromatic ring have more antiproliferative ability than those with one substitution at 3'-position. Esterification of the carboxyl group appears to increase the antiproliferative ability.     

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.     

Retinoic acid stimulates peptide leukotriene-syntheses in rat basophilic leukemia-1 (RBL-1) cells

Overnight incubation of rat basophilic leukemia-1 (RBL-1) cells with retinoic acid enhanced calcium ionophore-stimulated syntheses of LTC4 by more than 28-times (from 5.91 +/- 0.31 to 168.31 +/- 22.66 ng/2.10(6) cells) nd LTD4 by more than 7-times (from 5.27 +/- 0.12 to 39.38 +/- 14.89 ng/2.10(6) cells). The stimulatory action first appeared after a 10 h incubation with retinoic acid and was completely abolished by concomitant presence of low concentration cycloheximide (0.5 micrograms/ml) in the medium. However, LTB4 synthesis was dose-dependently inhibited by incubation with retinoic acid. Reduced form of glutathione significantly increased the synthesis of LTC4, but showed no action on the syntheses of LTD4 or LTB4. These results indicate a new enzyme synthesis of LTC4 synthetase.     

Lack of biological activity of physiological metabolites of all-trans-retinoic acid on vaginal epithelial differentiation

It has been of interest to determine whether the metabolites of physiological doses of retinoic acid represent active forms of vitamin A. Previous work (Biochem. J. 206, 33-41, 1982) studied the metabolites produced from 2-micrograms doses of all-trans-retinoic acid in the vitamin A-deficient rat. Four major metabolites common to all of the tissues studied were discovered. In the present work, three of these metabolites are isolated from vitamin A-deficient rats given physiological doses (5 micrograms) of all-trans-retinoic acid and from vitamin A-sufficient rats given high doses (1 mg) of all-trans-retinoic acid. Cochromatography on anion-exchange and reverse-phase high-performance liquid chromatography showed that metabolites resulting from high doses of retinoic acid contained the metabolites generated from physiological doses of retinoic acid. Quantities of these metabolites were isolated, purified, and tested for their epithelial-differentiating activity in the vitamin A-deficient rat vagina. The metabolites were inactive at all dose levels tested. These metabolites have less than 10% the biological activity of all-trans-retinoic acid. Therefore, these metabolites appear to be products of the inactivation of all-trans-retinoic acid. Based upon these and previous data, it seems likely that all-trans-retinoic acid or its beta-glucuronide derivative is the most likely active form of vitamin A in the maintenance of normal epithelial differentiation.     

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1

Glutathione transferase P1-1 is over expressed in some cancer cells and contributes to detoxification of anticancer drugs, leading to drug-resistant tumors. The inhibition of human recombinant GSTP1-1 by natural plant products was investigated using 10 compounds isolated from plants indigenous to Southern and Central Africa. Monochlorobimane and 1-chloro-2,4-dinitrobenzene were used to determine GST activity. Each test compound was screened at 33 and 100 µM. Isofuranonapthoquinone (1) (from Bulbine frutescens) showed 68% inhibition at 33 µM, and sesquiterpene lactone (2) (from Dicoma anomala) showed 75% inhibition at 33 μM. The IC₅₀ value of 1 was 6.8 μM. The mode of inhibition was mixed, partial (G site) and noncompetitive (H site) with K_i values of 8.8 and 0.21 µM, respectively. Sesquiterpene 2 did not inhibit the CDNB reaction. Therefore, isofuranonapthoquinone 1 needs further investigations in vivo because of its potent inhibition of GSTP1-1 in vitro.     

Inhibition of glutathione transferase pi from human placenta by 1-chloro-2,4-dinitrobenzene occurs because of covalent reaction with cysteine 47.

Human placenta glutathione transferase pi is irreversibly inhibited when incubated with 1-chloro-2,4-dinitrobenzene (CDNB) in the absence of the cosubstrate glutathione. The enzyme is protected against CDNB inactivation by the presence of S-methylglutathione and glutathione. The kinetics of inactivation is pseudo-first-order with k(obs) = 0.04 min-1 when 44 microM enzyme is incubated in presence of 1 mM CDNB at pH 6.5. The inhibition is saturable with respect to the CDNB concentration and the enzyme-CDNB complex shows a K(i) = 2.7 mM. Concomitant to the inhibition process is formation of an absorption band at 340 nm. After trypsin digestion and HPLC analysis, the CDNB-reacted enzyme gives a single peptide absorbing at 340 nm. Automated Edman degradation of this peptide indicates cysteine 47 to be the residue alkylated by CDNB.