Human Pi Class Glutathione S-transferase Research Articles

The diet, prostate inflammation, and the development of prostate cancer.

Evidence that somatic inactivation of GSTP1, encoding the human pi-class glutathione S-transferase, may initiate prostatic carcinogenesis is reviewed along with epidemiological evidence implicating several environment and lifestyle factors, including the diet and sexually transmitted diseases, as prostate cancer risk factors. An integrated model is presented featuring GSTPI function as a 'caretaker' gene during the pathogenesis of prostate cancer, in which the early loss of GSTPI activity renders prostate cells vulnerable to genome damage associated with chronic prostatic inflammation and repeated exposure to carcinogens. The model predicts that the critical prostate carcinogens will be those that are substrates for GSTP1 detoxification and are associated with high prostate cancer risk diet and lifestyle habits.

Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli.

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.

The ligandin (non-substrate) binding site of human pi class glutathione transferase is located in the electrophile binding site (H-site)

Glutathione S-transferases (GSTs) play a pivotal role in the detoxification of foreign chemicals and toxic metabolites. They were originally termed ligandins because of their ability to bind large molecules (molecular masses >400 Da), possibly for storage and transport roles. The location of the ligandin site in mammalian GSTs is still uncertain despite numerous studies in recent years. Here we show by X-ray crystallography that the ligandin binding site in human pi class GST P1-1 occupies part of one of the substrate binding sites. This work has been extended to the determination of a number of enzyme complex crystal structures which show that very large ligands are readily accommodated into this substrate binding site and in all, but one case, causes no significant movement of protein side-chains. Some of these molecules make use of a hitherto undescribed binding site located in a surface pocket of the enzyme. This site is conserved in most, but not all, classes of GSTs suggesting it may play an important functional role.

Genomic Cloning of hGSTP1*C, an Allelic Human Pi Class Glutathione S-Transferase Gene Variant and Functional Characterization of Its Retinoic Acid Response Elements

The complete hGSTP1*C, consisting of 7 exons and 6 introns contained in 3116 base pairs, was isolated from a cosmid genomic library of a glioblastoma multiforme cell line. Although the promoter of hGSTP1*C was identical to that of the previously reported GST-Pi gene, several of its structural features had not been previously described. These include several nucleotide transitions and transversions. Transitions of A --> G at +1404 and C --> T at +2294 in exons 5 and 6, respectively, changed codons Ile104 to Val104 and Ala113 to Val113. The gene also contained a guanine insertion at +51 in the insulin response element in intron 1 and six tandem repeats and one palindromic retinoic acid response element (RARE) consensus half-sites, A(G)GG(T)TC(G)A in intron 5. Retinoic acid (RA) treatment increased GST-Pi gene expression significantly in MGR3 cells. GST-Pi gene constructs with and without RARE deletion were used to show the RARE requirement for GST-Pi gene induction by RA. The isolation of the hGSTP1*C gene and the evidence that it contains functional RAREs should contribute to a better understanding of the molecular regulation of the GST-Pi gene in human cells.

The structures of human glutathione transferase P1-1 in complex with glutathione and various inhibitors at high resolution

The human pi-class glutathione S-transferase (hGST P1-1) is a target for structure-based inhibitor design with the aim of developing drugs that could be used as adjuvants in chemotherapeutic treatment. Here we present seven crystal structures of the enzyme in complex with substrate (glutathione) and two inhibitors ( S-hexyl glutathione and γ-glutamyl-( S-benzyl)cysteinyl- d-phenylglycine). The binding of the modified glutathione inhibitor, γ-glutamyl-( S-benzyl)cysteinyl- d-phenylglycine, has been characterized with the phenyl group stacking against the benzyl moiety of the inhibitor and making interactions with the active-site residues Phe8 and Trp38. The structure provides an explanation as to why this compound inhibits the pi-class GST much better than the other GST classes. The structure of the enzyme in complex with glutathione has been determined to high resolution (1.9 to 2.2 Å) in three different crystal forms and at two different temperatures (100 and 288 K). In one crystal form, the direct hydrogen-bonding interaction between the hydroxyl group of Tyr7, a residue involved in catalysis, and the thiol group of the substrate, glutathione, is broken and replaced by a water molecule that mediates the interaction. The hydrogen-bonding partner of the hydroxyl group of Tyr108, another residue implicated in the catalysis, is space-group dependent. A high-resolution (2.0 Å) structure of the enzyme in complex with S-hexyl glutathione in a new crystal form is presented. The enzyme-inhibitor complexes show that the binding of ligand into the electrophilic binding site does not lead to any conformational changes of the protein.

The human pi-class glutathione S-transferase: a target for structure-based drug design

The human pi-class glutathione S-transferase: a target for structure-based drug design

Functional Characterization of the Transcription Silencer Element Located within the Human Pi Class Glutathione S-Transferase Promoter

We have previously demonstrated enhanced transcriptional activity of the human Pi class glutathione S-transferase (GSTP1) promoter in a multidrug-resistant derivative (VCREMS) of the human mammary carcinoma cell line, MCF7 (Moffat, G. J., McLaren, A. W., and Wolf, C. R. (1994) J. Biol. Chem. 269, 16397-16402). Furthermore, we have identified an essential sequence (C1; -70 to -59) within the GSTP1 promoter that bound a Jun-Fos heterodimer in VCREMS but not in MCF7 cells. These present studies have examined the negative regulatory element (-105 to -86), which acted to suppress GSTP1 transcription in MCF7 cells. Mutational analysis of this silencer element further defined the repressor binding site to be located between nucleotides -97 and -90. In vitro DNA binding assays suggested that the repressor exerted its action by causing displacement of the essential non-AP-1-like MCF7 C1 complex. However, the addition of MCF7 nuclear extract did not disrupt binding of the VCREMS Jun-Fos C1 complex to the GSTP1 promoter. Furthermore, upstream insertion of the GSTP1 silencer element failed to inhibit activity of a heterologous promoter in MCF7 cells. These results highlighted the cell and promoter specificity of the GSTP1 transcriptional repressor and implicated a functional requirement for contact between the repressor and C1 complex. In this regard, the introduction of half-helical turns between the silencer and the C1 element abrogated repressor activity, thus leading to the hypothesis that a direct interaction between the repressor and C1 complex was required to suppress GSTP1 transcription. Moreover, these findings suggest that cell-specific differences in the composition of the C1 nuclear complex may dictate repressor activity.

Sp1-mediated Transcriptional Activation of the Human Pi Class Glutathione S-Transferase Promoter

Previous studies in this laboratory have identified an essential AP-1 recognition sequence (C1 region; -69 to -63) in th human Pi class glutathione s-transferase (GSTP1) promoter and a negatively acting regulatory element (-105 to -86) that acts to suppress GSTP1 transcription in the human mammary carcinoma cell line, MCF7 (1). The data presented here further delineate the functional characteristics of the GSTP1 promoter by examining the significance of two potential binding sites for the transcription factor, Sp1 (-57 to -49 and -47 to -39). The introduction of mutations within these Sp1-like elements and the use of Sp1 antisera in electrophoretic mobility shift assays demonstrated that Sp1 was bound to this region of the GSTP1 promoter in three different cell lines, MCF7, VCREMS, and EJ. Moreover, these in vitro studies indicated that only one of the two putative Sp1 response elements was utilized. Transient transfection assays using GSTP1 promoter constructs that incorporated mutations of the Sp1 elements clearly demonstrated that binding of Sp1 to the GSTP1 promoter was absolutely required for optimal levels of GSTP1 transcription. In particular, disruption of the distal Sp1 recognition motif (-57 to -49) markedly reduced GSTP1 promoter activity in each cell line, thus indicating preferential binding of Sp1 to the distal site. However, insertion of the repressor binding site (-105 to -86) into these constructs suggested that Sp1 was not involved in mediating the suppressive effects of the GSTP1 transcriptional repressor in MCF7 cells, because inhibition of Sp1 binding did not alleviate repressor activity. Therefore, these studies provide strong evidence that Sp1 plays a central role in regulating basal levels of GSTP1 transcription.

Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase.

Expression of cloned genes in prokaryotes such as Escherichia coli is a widely used technique in both basic research and biotechnology. Despite the availability of several E. coli expression vector systems, adequate levels of expression may not be achieved. Expressing proteins as fusions to the highly conserved eukaryotic protein ubiquitin has been reported by several investigators to enhance protein yield in both bacterial and eukaryotic systems. We have modified this technique by the co-expression in E. coli of a ubiquitin-fusion protein and the Saccharomyces cerevisiae ubiquitin-specific protease Ubp2. This allows the co-translational cleavage of engineered ubiquitin-fusion proteins expressed in E. coli. This system was used to express a human Pi class glutathione S-transferase (GST) GSTP1 as well as two mutant GSTP1 derivatives, Trp39-->Cys and Gln52-->Glu. The yield of these enzymes was improved 40-fold by using the ubiquitin-fusion/co-translational cleavage technique, and no uncleaved product was detected. The Trp39-->Cys mutant was totally devoid of GST activity, while the activity of the Gln52-->Glu mutant was reduced to 6% of wild-type GSTP1-1. As both of the mutated residues map within the glutathione-binding site, the reduced GST activity is consistent with a marked reduction in glutathione binding ability.

Involvement of Jun and Fos proteins in regulating transcriptional activation of the human pi class glutathione S-transferase gene in multidrug-resistant MCF7 breast cancer cells.

Elevated levels of the human pi class glutathione S-transferase (GSTP1-1) have been implicated in the development of antineoplastic drug resistance. Using GSTP1 promoter deletion constructs we have shown that enhanced GSTP1 transcription (up to 18-fold) is the predominant mechanism responsible for increased GSTP1-1 levels in a multidrug resistant derivative (VCREMS) of the human mammary carcinoma cell line MCF7. Furthermore, disruption of a putative AP-1 response element within the GSTP1 promoter (nucleotides -69 to -63) abrogated GSTP1 transcription in both cell lines. In addition, band shift assays demonstrated binding of a VCREMS nuclear complex to the promoter region C1 (-73 to -54) which could be competed for by a DNA fragment containing a known AP-1 binding site from the human collagenase promoter. However, no such competition was observed for the major MCF7 C1 complex. The role of a Fos-Jun-like complex in regulating GSTP1 transcription in VCREMS cells was further emphasized by the introduction of point mutations within the C1 region which were known to inhibit AP-1 binding and the interaction of antisera raised against human c-Jun and c-Fos with the major C1 complex in VCREMS cells. These studies therefore highlight cell-specific differences in the binding pattern of Jun and Fos proteins to the GSTP1 promoter which are likely to play an important role in regulating transcriptional activation of the GSTP1 gene in drug-resistant breast cancer cells.

Protection by transfected glutathione S-transferase isozymes against carcinogen-induced alkylation of cellular macromolecules in human MCF-7 cells.

Increased expression of glutathione S-transferase (GST) isozymes has been correlated with development of resistance both to cytotoxic anticancer agents and to genotoxic carcinogens. While most anticancer agents are poor GST substrates, the model alkylating carcinogen 4-nitroquinoline-1-oxide (NQO) is a good substrate for human pi class GST (hGSTP1-1) and murine GST mu-1 (mGSTM1-1), but not human GST alpha-2 (hGSTA2-2). We investigated whether expression of these GST isozymes in stably transfected clonal cell lines could protect against the genotoxic and cytotoxic effects of NQO. Compared to parental MCF-7 or pSV2neotransfected control cell lines, covalent labeling of total cellular macromolecules by [3H]NQO (0.1-1.0 mM) was reduced by 70% and 92% in hGSTP1-1- and mGSTM1-1-transfected cell lines, respectively, but was not affected in the hGSTA2-2 expressing line. The observed protection was closely correlated with the relative specific activity of each cell line for conjugation of NQO by the transfected GST isozymes and this protection was reversible by pretreatment of cells with the GST inhibitor ethacrynic acid. Similar results were obtained when covalent labeling of total cellular nucleic acid or DNA was measured. However, clonogenic survival assays indicated that the sensitivity of these cell lines to the cytotoxic effects of NQO was similar for the control and GST-transfected MCF-7 cell lines. Thus, while expression of hGSTP1-1 and mGSTM1-1 (but not hGSTA2-2) was highly protective against alkylation of cellular macromolecules by NQO, this protection was not effective against cytotoxicity induced by NQO as measured by clonogenic assay. These results indicate that expression of GST isozymes can protect differentially against the acute genotoxic and potentially mutagenic effects, as compared to the cytotoxic effects, of electrophiles that are detoxified by glutathione conjugation.

Benastatins C and D, new inhibitors of glutathione S-transferase, produced by Streptomyces sp. MI384-DF12. Production, isolation, structure determination and biological activities.

Benastatin C, a new member of the benastatins, has been isolated from the culture broth of Streptomyces sp. MI384-DF12. The structure of benastatin C was elucidated as 2-decarboxy-benastatin A by NMR studies. Benastatin D, 2-decarboxybenastatin B, was derived from benastatin B. Benastatins C and D showed inhibitory activities against human pi class glutathione S-transferase (GST pi) and excellent stimulatory activities on the murine lymphocyte blastogenesis in vitro.

Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases.

1. The diuretic drug ethacrynic acid (EA) is a potent reversible inhibitor of rat and human glutathione S-transferases (GST), with I50-values (microM) of 4.6-6.0, 0.3-1.9 and 3.3-4.8 for alpha, mu and pi-class, respectively. 2. The reversible inhibition by the glutathione conjugate of EA is even stronger for alpha and mu-class, with I50-values (microM) of 0.8-2.8 and < 0.1-1.2, respectively, while the I50 for the pi-class is 11. 3. Inhibition of rat and human pi-class GST also occurs by covalent binding of ethacrynic acid. 14C-ethacrynic acid, 0.8 nmol EA per nmol pi-class GST could be incorporated, resulting in 65-93% inhibition of the catalytic activity. 4. Owing to the chemical nature of the covalent binding (Michael addition), this reaction should be reversible. Indeed, full restoration of the catalytic activity of GST P1-1 inactivated by covalently-bound EA was reached in about 125 h by incubation with an excess of glutathione. 5. EA has been used to inhibit GST in biological systems. The reversible covalent binding may very well play a role in the observed inhibition of GST by EA in vivo.

The major glutathione S-transferase in salmonid fish livers is homologous to the mammalian pi-class GST

1. 1. The hepatic glutathione S-transferase (GST) isoenzymes were isolated and characterized from salmon, sea trout and rainbow trout. 2. 2. In all three species the predominant GST expressed comprised subunits of M r 24,800. These subunits each co-migrated with the rat pi-class Yf polypeptide during SDS/polyacrylamide gel electrophoresis. 3. 3. Western blotting experiments demonstrated immunochemical cross-reactivity between the major salmonid and the rat pi-class GSTs. 4. 4. The salmon GST of subunit M r 24,800 was digested with cyanogen bromide and the peptides, once purified by reverse-phase HPLC, were subjected to automated amino acid sequencing. 5. 5. Over the region sequenced, the salmon GST possessed about 65% homology with the rat and human pi-class GST.