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)

Recent epidemiological studies proposed that the glutathione S-transferase (GST) M1-null genotype may contribute to diseases associated with oxidative stress. The genetic polymorphism exhibited by the GSTM1 may be an important factor in risk toward oxidant chemicals. In this study, we investigated the effect of GSTM1-null genotype in lymphocyte and oxidative stress-dependent inhibition of platelet aggregation. To determine whether GSTM1 deficiency is a genetic determinant of cell toxicity toward oxidant chemicals, lymphocytes were incubated in vitro with low levels of benzo(a)pyrene (BaP), cumene hydroperoxide (CumOOH), or trans-stilbene oxide that do not decrease cell viability, and were assessed for oxidative damage and for the lymphocyte-dependent inhibition of platelet response. Malondialdehyde and carbonyl levels, and the oxidation of cisparinaric acid, were used as biomarkers of oxidative stress in lymphocytes. Following stimulation by BaP or CumOOH, when peroxidation-dependent changes in these parameters were compared between the GSTM1-null genotype and the positive genotype, no significant differences were found between the two genotypes. On the other hand, preincubation of the lymphocytes with BaP or CumOOH attenuated their inhibitory action on ADP-induced platelet aggregation. However, our results indicate that lymphocytes of individuals with the GSTM1-null genotype have greater inhibitory activity on platelet function after exposure to BaP, but not CumOOH, although they are not more susceptible to in vitro oxidative stress.
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PMID:Inhibition of platelet function by GSTM1-null human peripheral lymphocytes exposed to benzo(a)pyrene-induced challenge. 1120 Oct 55

The transcription factor NF-kappaB regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis. IkappaB inhibitory subunits integrate NF-kappaB activation signals through phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for IkappaB-alpha N-terminal domain interactors and therefore potential NF-kappaB regulators. An interaction of IkappaB-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IkappaB-alpha and associated p65 NF-kappaB in the mitochondrial intermembrane space. IkappaB-alpha.NF-kappaB appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor. These data suggest that the mitochondrial IkappaB-alpha.NF-kappaB pool participates in the regulation of apoptosis.
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PMID:Ikappa b-alpha, the NF-kappa B inhibitory subunit, interacts with ANT, the mitochondrial ATP/ADP translocator. 1128 11

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.
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PMID:Adenylate kinase as a virulence factor of Pseudomonas aeruginosa. 1134 42

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.
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PMID:Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding. 1160 53

The binary Clostridium botulinum C2 toxin is composed of the enzyme component C2I and the binding component C2II, which are individual and non-linked proteins. Activated C2IIa mediates cell binding and translocation of C2I into the cytoplasm. C2I ADP-ribosylates G-actin at Arg-177 to depolymerize actin filaments. A fusion toxin containing the N-terminal domain of C2I (residues 1-225) transports C3 ADP-ribosyltransferase from Clostridium limosum into cells (Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K. (1998) Infect. Immun. 66, 1364-1369). We characterized the adaptor function of C2I and its interaction with C2IIa. The fusion toxin GST-C2I(1-225)-C3 was efficiently transported by C2IIa, indicating that C2IIa translocates proteins into the cytosol even when the C2I(1-225) adaptor was positioned in the middle of a fusion protein. Amino acid residues 1-87 of C2I were sufficient for interaction with C2IIa and for translocation of C2I fusion toxins into HeLa cells. Residues 1-87 were the minimal part of C2I to bind to C2IIa on the cell surface, as detected by fluorescence-activated cytometry. An excess of C2I(1-87) (but not of further truncated C2I fragments) competed with Alexa488-labeled C2I for binding to C2IIa. Also, the fragment C2I(30-431) and the fusion toxin C2I(30-225)-C3 competed with C2I-Alexa488 for binding to C2IIa. C2I(30-225)-C3 did not induce cytotoxic effects on cells when applied together with C2IIa, indicating that amino acid residues 1-29 are involved in translocation of C2I but are not absolutely essential for binding to C2IIa.
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PMID:The binary Clostridium botulinum C2 toxin as a protein delivery system: identification of the minimal protein region necessary for interaction of toxin components. 1174 86

RhoA, -B, and -C are ADP-ribosylated and biologically inactivated by Clostridium botulinum C3 exoenzyme and related C3-like transferases. We report that RalA GTPase, which is not ADP-ribosylated by C3, inhibits ADP-ribosylation of RhoA by C3 from C. botulinum (C3bot), Clostridium limosum (C3lim), and Bacillus cereus (C3cer) but not from Staphylococcus aureus (C3stau) in human platelet membranes and rat brain lysate. Inhibition by RalA occurs with the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of RalA and is overcome by increasing concentrations of C3. A direct interaction of RalA with C3 was verified by precipitation of the transferase with GST-RalA-Sepharose. The affinity constant (K(d)) of the binding of RalA to C3lim was 12 nm as determined by fluorescence titration. RalA increased the NAD glycohydrolase activity of C3bot by about 5-fold. Although RalA had no effect on glucosylation of Rho GTPases by Clostridium difficile toxin B, C3bot and C3lim inhibited glucosylation of RalA by Clostridium sordellii lethal toxin. Furthermore, C3bot decreased activation of phospholipase D by RalA. The data indicate that several C3 exoenzymes directly interact with RalA without ADP-ribosylating the GTPase. The interaction is of high affinity and interferes with essential functions of C3 and RalA.
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PMID:Interaction of the Rho-ADP-ribosylating C3 exoenzyme with RalA. 1184 34

The release of cytochrome c from mitochondria is a critical step during apoptosis. In order to study this process, we have used a synthetic compound, MT-21, that is able to initiate release of cytochrome c from isolated mitochondria. We demonstrate that MT-21 significantly inhibits ADP transport activity in mitochondria and reduces binding of the adenine nucleotide translocase (ANT) to a phenylarsine oxide affinity matrix. These results suggest that ANT, one of the components of the mitochondrial permeability transition (PT) pore, is the molecular target for MT-21. In agreement with this, the MT-21-induced cytochrome c release was effectively inhibited in the presence of ANT ligands, and MT-21 could dissociate ANT from a complex with a glutathione S-transferase-cyclophilin D fusion protein. Interestingly, we also found that specific inhibitors of ANT such as MT-21 and atractyloside could induce cytochrome c release without mitochondrial swelling and that this event was highly dependent on the presence of Mg(2+). These results suggest that although ANT resides in the mitochondrial inner membrane, specific ANT inhibitors can induce cytochrome c release without having an effect on inner membrane permeability. Therefore, MT-21 can be a powerful tool for studying the mechanism of PT-independent cytochrome c release from mitochondria.
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PMID:A novel adenine nucleotide translocase inhibitor, MT-21, induces cytochrome c release by a mitochondrial permeability transition-independent mechanism. 1206 61

Opening of the mitochondrial permeability transition pore (MPTP) is sensitized to [Ca(2+)] by oxidative stress (diamide) and phenylarsine oxide (PAO). We have proposed that both agents cross-link two thiol groups on the adenine nucleotide translocase (ANT) involved in ADP and cyclophilin-D (CyP-D) binding. Here, we demonstrate that blocking Cys(160) with 80 microM eosin 5-maleimide (EMA) or 500 microM N-ethylmaleimide (NEM) greatly decreased ADP inhibition of the MPTP. The ability of diamide, but not PAO, to block ADP inhibition of the MPTP was antagonized by treatment of mitochondria with 50 microM NEM to alkylate matrix glutathione. Binding of detergent-solubilized ANT to a PAO-affinity matrix was prevented by pre-treatment of mitochondria with diamide, EMA or PAO, but not NEM. EMA binding to the ANT in submitochondrial particles (SMPs) was prevented by pre-treatment of mitochondria with either PAO or diamide, implying that both agents modify Cys(160). Diamide and PAO pre-treatments also inhibited binding of solubilized ANT to a glutathione S-transferase-CyP-D affinity column, both effects being blocked by 100 microM EMA. Intermolecular cross-linking of adjacent ANT molecules via Cys(57) by copper phenanthroline treatment of SMPs was abolished by pre-treatment of mitochondria with diamide and PAO, but not with EMA. Our data suggest that PAO and diamide cause intramolecular cross-linking between Cys(160) and Cys(257) directly (not antagonized by 50 microM NEM) or using glutathione (antagonized by 50 microM NEM) respectively. This cross-linking stabilizes the "c" conformation of the ANT, reducing the reactivity of Cys(57), while enhancing CyP-D binding to the ANT and antagonizing ADP binding. The two effects together greatly sensitize the MPTP to [Ca(2+)].
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PMID:Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. 1214 99

To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro. The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1. Expression of fusion protein was directed by the tac promoter. The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction. The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography. GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained. The GST-KGDX was expressed in E. coli to a level of 48.02% of total cellular protein. GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01). In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa. Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST. It is concluded that the GST-KGDX fusion protein can be produced by E. coli and used as an antiplatelet agent.
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PMID:[Expression and purification of recombinant glycoprotein (GP) IIb/IIIa receptor antagonists]. 1251 16

The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd approximately 175 microM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd approximately 3 microM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in approximately 8-fold tighter binding of PDK2 to GST-L2red, which was approximately 300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound approximately 18 PDK2 dimers with a Kd similar to GST-L2. E2.E1 bound more PDK2 (approximately 27.6) than E2 with approximately 2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2.E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5'-(beta,gamma-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2.E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed "hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate.
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PMID:Facilitated interaction between the pyruvate dehydrogenase kinase isoform 2 and the dihydrolipoyl acetyltransferase. 1281 49


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