Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation. The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells. Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation. This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, do not cause transformation. In cells overexpressing active hGSTA4-4, expression of transforming growth factor beta1, cyclin-dependent kinase 2, protein kinase C betaII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated. These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events.
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PMID:Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells. 1509 8

The cellular production of 4-hydroxy-2-nonenal (HNE), a product of endogenous lipid peroxidation, constitutes a genotoxic risk factor for carcinogenesis. Our previous studies have shown that human HT29 colon cells developed resistance toward HNE injury after treatment with butyrate, a diet-associated gut fermentation product. This resistance was attributed to the induction of certain glutathione S-transferases (hGSTP1-1, hGSTM2-2, and hGSTA1-1) and also for the tripeptide glutathione (GSH) synthesizing enzymes. In the present study, we have investigated in HT29 cells whether hGSTA4-4, which has a high substrate specificity for HNE, was also inducible by butyrate and, thus, could contribute to the previously observed chemoresistance. In addition, we investigated if cellular depletion of GSH by L-buthionine-S,R-sulfoximine (BSO) enhances chemosensitivity to HNE injury in HT29 cells. Incubation of HT29 cells with butyrate (2-4 mM) significantly elicited a 1.8 to 3-fold upregulation of steady state hGSTA4 mRNA over 8-24 h after treatment. Moreover, 4 mM butyrate tended to increase hGSTA4-4 protein concentrations. Incubation with 100 microM BSO decreased cellular GSH levels by 77% without significant changes in cell viability. Associated with this was a 2-fold higher level of HNE-induced DNA damage as measured by the comet assay. Collectively, the results of this study and our previous work indicate that the genotoxicity of HNE is highly dependent on cellular GSH status and those GSTs that contribute toward HNE conjugation, including hGSTA4-4. Since HNE contributes to colon carcinogenesis, the favorable modulation of the GSH/GST system by butyrate may contribute to chemoprevention and reduction of the risks.
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PMID:Genotoxicity of 4-hydroxy-2-nonenal in human colon tumor cells is associated with cellular levels of glutathione and the modulation of glutathione S-transferase A4 expression by butyrate. 1582 14

The human alpha class glutathione S-transferases (GSTs) consist of 5 genes, hGSTA1-hGSTA5, and 7 pseudogenes on chromosome 6p12.1-6p12.2. hGSTA1-hGSTA4 have been well characterized as proteins, but hGSTA5 has not been detected as a gene product. hGSTA1-1 (and to a lesser extent hGSTA2-2) catalyzes the GSH-dependent detoxification of carcinogenic metabolites of environmental pollutants and tobacco smoke (e.g., polycyclic aromatic hydrocarbon diolepoxides) and several alkylating chemotherapeutic agents and has peroxidase activity toward fatty acid hydroperoxides (FA-OOH) and phosphatidyl FA-OOH. hGSTA3-3 has high activity for the GSH-dependent Delta(5)-Delta(4) isomerization of steroids, and hGSTA4-4 has high activity for the GSH conjugation of 4-hydroxynonenal. hGSTA4 is expressed in many tissues; hGSTA1-1 and hGSTA2-2 are expressed at high levels in liver, intestine, kidney, adrenal gland, and testis; and hGSTA3 is expressed in steroidogenic tissues. Functional, allelic, single nucleotide polymorphisms occur in an SP1-binding element of hGSTA1 and in the coding regions of hGSTA2 and hGSTA3. The main effects of these polymorphisms are the low hepatic expression of hGSTA1 in individuals homozygous for hGSTA1*B and the low specific activity of the hGSTA2E-2E variant toward FA-OOH. These properties suggest that alpha class GSTs will be involved in susceptibility to diseases with an environmental component (such as cancer, asthma, and cardiovascular disease) and in response to chemotherapy. Although hGSTM1, hGSTT1, and hGSTP1 have been associated with such diseases (on the basis of genetic polymorphisms as indicators of expression), alpha class GSTs have been little studied in this respect. Nevertheless, hGSTA1*B has been associated with increased susceptibility to colorectal cancer and with increased efficacy of chemotherapy for breast cancer. Methods for identification and quantitation of human alpha class GST protein, mRNA, and genotype are reviewed, and the potential for GST-alpha in plasma to be used as a marker for hepatic expression and induction is discussed.
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PMID:Human alpha class glutathione S-transferases: genetic polymorphism, expression, and susceptibility to disease. 1639 77

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.
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PMID:Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria. 1661 95

Epidemiological studies have shown that ingestion of isoflavone-rich soy products is associated with a reduced risk for the development of breast cancer. In the present study, we investigated the hypothesis that genistein modulates the expression of glutathione S-transferases (GSTs) in human breast cells, thus conferring protection towards genotoxic carcinogens which are GST substrates. Our approach was to use human mammary cell lines MCF-10A and MCF-7 as models for non-neoplastic and neoplastic epithelial breast cells, respectively. MCF-10A cells expressed hGSTA1/2, hGSTA4-4, hGSTM1-1 and hGSTP1-1 proteins, but not hGSTM2-2. In contrast, MCF-7 cells only marginally expressed hGSTA1/2, hGSTA4-4 and hGSTM1-1. Concordant to the protein expression, the hGSTA4 and hGSTP1 mRNA expression was higher in the non-neoplastic cell line. Exposure to genistein significantly increased hGSTP1 mRNA (2.3-fold), hGSTP1-1 protein levels (3.1-fold), GST catalytic activity (4.7-fold) and intracellular glutathione concentrations (1.4-fold) in MCF-10A cells, whereas no effects were observed on GST expression or glutathione concentrations in MCF-7 cells. Preincubation of MCF-10A cells with genistein decreased the extent of DNA damage by 4-hydroxy-2-nonenal (150 microM) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (50 microM), compounds readily detoxified by hGSTA4-4 and hGSTP1-1. In conclusion, genistein pretreatment protects non-neoplastic mammary cells from certain carcinogens that are detoxified by GSTs, suggesting that dietary-mediated induction of GSTs may be a mechanism contributing to prevention against genotoxic injury in the aetiology of breast cancer.
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PMID:Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system. 1706 99

Tolerance to clinically important organic nitrates such as nitroglycerin (NTG) has been experimentally related to endothelial dysfunction and vascular oxidative stress. Anti-oxidant enzymes such as the glutathione-S-transferases GSTs) could potentially play a protective role in NTG tolerance. Our previous work showed that an alpha-class glutathione-S-transferase (GSTA4-4) defends against oxidative damage in the vascular wall; therefore, we asked whether overexpression of GSTA4-4 in endothelial cells and smooth muscle cells might alter the development of tolerance to NTG. Stable transfections of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, and human fetal aortic vascular smooth muscle cells (FLTR) with cDNA of hGSTA4-4 were established. MTT cytotoxicity, apoptosis, nitric oxide (NO) synthases, both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) and cyclic guanosine mono-phosphate (cGMP) were measured. Endothelial cells overexpressing mGSTA4-4, and smooth muscle cells overexpressing hGSTA4-4 were more resistant to cytotoxic injury by NTG, assessed at 24 h (p < 0.05). In both endothelial and smooth muscle cells, NTG-induced apoptosis was inhibited by GST overexpression. Following dosing in a relevant tolerance-inducing NTG protocol, we found that GSTA4-4-overexpressing cells demonstrated significant downregulation of NOS enzymes; NO release, unchanged by the tolerance protocol in both wild-type and vector-transfected cells, was augmented in GST-overexpressing cells (p < 0.01); cGMP levels in control cells fell, whereas it rose in GSTA4-4-overexpressing cells (p < 0.05). Our results demonstrate that overexpression of GST isozymes can protect endothelial cells and smooth muscle cells against oxidative stress associated with NTG, and markedly alter cellular responses to repeated doses, or tolerance. By manipulating GSTs, physiological tolerance to NTG may be diminished or eliminated.
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PMID:Manipulating glutathione-S-transferases may prevent the development of tolerance to nitroglycerin. 1730 20

cis-6-(2-Acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG) are thiopurine prodrugs provisionally inactivated by an alpha,beta-unsaturated substituent on the sulfur of the parental thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). The active thiopurines are liberated intracellularly by glutathione (GSH) in reactions catalyzed by glutathione transferases (GSTs) (EC 2.5.1.18). Catalytic activities of 13 human GSTs representing seven distinct classes of soluble GSTs have been determined. The bioactivation of cAVTP and tAVTG occurs via a transient addition of GSH to the activated double bond of the S-substituent of the prodrug, followed by elimination of the thiopurine. The first of these consecutive reactions is rate-limiting for thiopurine release, but GST-activation of this first addition is shifting the rate limitation to the subsequent elimination. Highly active GSTs reveal the transient intermediate, which is detectable by UV spectroscopy and HPLC analysis. LC/MS analysis of the reaction products demonstrates that the primary GSH conjugate, 4-glutathionylbuten-2-one, can react with a second GSH molecule to form the 4-(bis-glutathionyl)butan-2-one. GST M1-1 and GST A4-4 were the most efficient enzymes with tAVTG, and GST M1-1 and GST M2-2 had highest activity with cAVTP. The highly efficient GST M1-1 is polymorphic and is absent in approximately half of the human population. GST P1-1, which is overexpressed in many cancer cells, had no detectable activity with cAVTP and only minor activity with tAVTG. Other GST-activated prodrugs have targeted GST P1-1-expressing cancer cells. Tumors expressing high levels of GST M1-1 or GST A4-4 can be predicted to be particularly vulnerable to chemotherapy with cAVTP or tAVTG.
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PMID:Human glutathione transferases catalyzing the bioactivation of anticancer thiopurine prodrugs. 1743 63

Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J = 0.00) to highly promiscuous (cruzain, J = 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J = 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes.
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PMID:A quantitative index of substrate promiscuity. 1808 10

Redesign of glutathione transferases (GSTs) has led to enzymes with remarkably enhanced catalytic properties. Exchange of substrate-binding residues in GST A1-1 created a GST A4-4 mimic, called GIMFhelix, with >300-fold improved activity with nonenal and suppressed activity with other substrates. In the present investigation GIMFhelix was compared with the naturally-evolved GSTs A1-1 and A4-4 by determining catalytic efficiencies with nine alternative substrates. The enzymes can be represented by vectors in multidimensional substrate-activity space, and the vectors of GIMFhelix and GST A1-1, expressed in kcat/Km values for the alternative substrates, are essentially orthogonal. By contrast, the vectors of GIMFhelix and GST A4-4 have approximately similar lengths and directions. The broad substrate acceptance of GST A1-1 contrasts with the high selectivity of GST A4-4 and GIMFhelix for alkenal substrates. Multivariate analysis demonstrated that among the diverse substrates used, nonenal, cumene hydroperoxide, and androstenedione are major determinants in the portrayal of the three enzyme variants. These GST substrates represent diverse chemistries of naturally occurring substrates undergoing Michael addition, hydroperoxide reduction, and steroid double-bond isomerization, respectively. In terms of function, GIMFhelix is a novel enzyme compared to its progenitor GST A1-1 in spite of 94% amino-acid sequence identity between the enzymes. The redesign of GST A1-1 into GIMFhelix therefore serves as an illustration of divergent evolution leading to novel enzymes by minor structural modifications in the active site. Notwithstanding low sequence identity (60%), GIMFhelix is functionally an isoenzyme of GST A4-4.
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PMID:Emergence of a novel highly specific and catalytically efficient enzyme from a naturally promiscuous glutathione transferase. 1870 75

In recent years, 4-hydroxy-2-nonenal (4-HNE) has emerged as an important second messenger in cell cycle signaling. Here, we demonstrate that 4-HNE induces signaling for apoptosis via both the Fas-mediated extrinsic and the p53-mediated intrinsic pathways in HepG2 cells. 4-HNE induces a Fas-mediated DISC independent apoptosis pathway by activating ASK1, JNK, and caspase-3. Parallel treatment of 4-HNE to HepG2 cells also induces apoptosis by the p53 pathway through activation of Bax, p21, JNK, and caspase-3. Exposure of HepG2 cells to 4-HNE leads to the activation of both Fas and Daxx, promotes the export of Daxx from the nucleus to cytoplasm, and facilitates Fas-Daxx binding. Depletion of Daxx by siRNA results in the potentiation of apoptosis, indicating that Fas-Daxx binding in fact is inhibitory to Fas-mediated apoptosis in cells. 4-HNE-induced translocation of Daxx is also accompanied by the activation and nuclear accumulation of HSF1 and up-regulation of heat shock protein Hsp70. All these effects of 4-HNE in cells can be attenuated by ectopic expression of hGSTA4-4, the isozyme of glutathione S-transferase with high activity for 4-HNE. Through immunoprecipitation and liquid chromatography-tandem mass spectrometry, we have demonstrated the covalent binding of 4-HNE to Daxx. We also demonstrate that 4-HNE modification induces phosphorylation of Daxx at Ser668 and Ser671 to facilitate its cytoplasmic export. These results indicate that while 4-HNE exhibits toxicity through several mechanisms, in parallel it evokes signaling for defense mechanisms to self-regulate its toxicity and can simultaneously affect multiple signaling pathways through its interactions with membrane receptors and transcription factors/repressors.
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PMID:Mechanisms of 4-hydroxy-2-nonenal induced pro- and anti-apoptotic signaling. 2056 32


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