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)

One early response to epidermal growth factor receptor (EGFR) activation is an increase in intracellular calcium. We have used surface plasmon resonance (SPR) to study real-time interactions between the intracellular juxtamembrane (JM) region of EGFR and calmodulin. The EGFR-JM (Met(644)-Phe(688)) was expressed as a GST fusion protein and immobilised on a sensor chip surface. Calmodulin specifically interacts with EGFR-JM in a calcium-dependent manner with a high on and high off rate. Chemical modification of EGFR-JM by using arginine-selective phenylglyoxal or deletion of the basic segment Arg(645)-Arg(657) inhibits the interaction. Phosphorylation of EGFR-JM by protein kinase C (PKC) or glutamate substitution of Thr(654) inhibits the interaction, suggesting that PKC phosphorylation electrostatically interferes with calmodulin binding to basic arginine residues. Calmodulin binding was also inhibited by suramin. Our results suggest that EGFR-JM is essential for epidermal growth factor (EGF)-mediated calcium-calmodulin signalling and for signal integration between other signalling pathways.
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PMID:Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance. 1235 6

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.
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PMID:Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation. 1247 Oct 40

A variant form of an Anopheles dirus glutathione S-transferase (GST), designated AdGSTD4-4, possesses a single amino acid change of leucine to arginine (Leu-103-Arg). Although residue 103 is outside of the active site, it has major effects on enzymic properties. To investigate these structural effects, site-directed mutagenesis was used to generate mutants by changing the non-polar leucine to alanine, glutamate, isoleucine, methionine, asparagine, or tyrosine. All of the recombinant GSTs showed approximately the same expression level at 25 degrees C. Several of the mutants lacked glutathione (GSH)-binding affinity but were purified by S-hexyl-GSH-based affinity chromatography. However the protein yields (70-fold lower), as well as the GST activity (100-fold lower), of Leu-103-Tyr and Leu-103-Arg purifications were surprisingly low and precluded the performance of kinetic experiments. Size-exclusion chromatography showed that both GSTs Leu-103-Tyr and Leu-103-Arg formed dimers. Using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH substrates to determine kinetic constants it was demonstrated that the other Leu-103 mutants possessed a greater K (m) towards GSH and a differing K (m) towards CDNB. The V (max) ranged from 44.7 to 87.0 micromol/min per mg (wild-type, 44.7 micromol/min per mg). Substrate-specificity studies showed different selectivity properties for each mutant. The structural residue Leu-103 affects the active site through H-bond and van-der-Waal contacts with six active-site residues in the GSH binding site. Changes in this interior core residue appear to disrupt internal packing, which affects active-site residues as well as residues at the subunit-subunit interface. Finally, the data suggest that Leu-103 is noteworthy as a sensitive residue in the GST structure that modulates enzyme activity as well as stability.
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PMID:A sensitive core region in the structure of glutathione S-transferases. 1270 68

Astrocytes have a higher antioxidant potential in comparison to neurons. Pathways associated with this selective advantage include the transcriptional regulation of antioxidant enzymes via the action of the Cap'n'Collar transcription factor Nrf2 at the antioxidant response element (ARE). Here we show that Nrf2 overexpression can reengineer neurons to express this glial pathway and enhance antioxidant gene expression. However, Nrf2-mediated protection from oxidative stress is conferred primarily by glia in mixed cultures. The antioxidant properties of Nrf2-overexpressing glia are more pronounced than those of neurons, and a relatively small number of these glia (< 1% of total cell number added) could protect fully cocultured naive neurons from oxidative glutamate toxicity associated with glutathione (GSH) depletion. Microarray and biochemical analyses indicate a coordinated upregulation of enzymes involved in GSH biosynthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase, and GSH synthase), use (glutathione S-transferase and glutathione reductase), and export (multidrug resistance protein 1) with Nrf2 overexpression, leading to an increase in both media and intracellular GSH. Selective inhibition of glial GSH synthesis and the supplementation of media GSH indicated that an Nrf2-dependent increase in glial GSH synthesis was both necessary and sufficient for the protection of neurons, respectively. Neuroprotection was not limited to overexpression of Nrf2, because activation of endogenous glial Nrf2 by the small molecule ARE inducer, tert-butylhydroquinone, also protected against oxidative glutamate toxicity.
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PMID:Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. 1271 47

Dynamic regulation of synaptic AMPA receptor localization underlies certain forms of synaptic plasticity and researchers are just beginning to identify molecules that may play a role in the synaptic delivery of glutamate receptors. One candidate is mLin-10, the mammalian homolog of the C. elegans receptor targeting protein LIN-10. Here, we investigated the role of mLin-10 in glutamate receptor trafficking. Cellular localization studies, in both whole brain and cultured neurons, revealed that mLin-10 is enriched in the trans-Golgi network and present in dendrites and spines--regions where protein sorting and synaptic delivery are known to occur. The specific localization of mLin-10 in Golgi is disrupted by a point mutation in an mLin-10 PDZ domain, indicating that a PDZ domain mediates this localization. Interactions between mLin-10 and glutamate receptors in both intracellular and synaptic membrane fractions were detected through biochemical assays. GST-pull down and co-immunoprecipitation experiments in heterologous cells delineated the protein domains required for interaction. These results demonstrated that glutamate receptors interact directly with mLin-10 through a PDZ domain-mediated mechanism. A PDZ point mutation enhances surface delivery of exogenous glutamate receptors in transfected neurons, suggesting that mLin-10 may regulate AMPA receptor trafficking in vivo.
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PMID:The PDZ domains of mLin-10 regulate its trans-Golgi network targeting and the surface expression of AMPA receptors. 1452 21

Dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) is an organophosphorus (OP) insecticide and acaricide extensively used to treat external parasitic infections of farmed fish. In previous studies we have demonstrated the importance of the glutathione (GSH) metabolism in the resistance of the European eel (Anguilla anguilla L.) to thiocarbamate herbicides. The present work studied the effects of the antioxidant and glutathione pro-drug N-acetyl-L-cysteine (NAC) on the survival of a natural population of A. anguilla exposed to a lethal concentration of dichlorvos, focusing on the glutathione metabolism and the enzyme activities of acetylcholinesterase (AChE) and caspase-3 as biomarkers of neurotoxicity and induction of apoptosis, respectively. Fish pre-treated with NAC (1 mmol kg(-1), i.p.) and exposed to 1.5 mg l(-1) (the 96-h LC85) of dichlorvos for 96 h in a static-renewal system achieved an increase of the GSH content, GSH/GSSG ratio, hepatic glutathione reductase (GR), glutathione S-transferase (GST), glutamate:cysteine ligase (GCL), and gamma-glutamyl transferase (gammaGT) activities, which ameliorated the glutathione loss and oxidation, and enzyme inactivation, caused by the OP pesticide. Although NAC-treated fish presented a higher survival and were two-fold less likely to die within the study period of 96 h, Cox proportional hazard models showed that hepatic GSH/GSSG ratio was the best explanatory variable related to survival. Hence, tolerance to a lethal concentration of dichlorvos can be explained by the individual capacity to maintain and improve the hepatic glutathione redox status. Impairment of the GSH/GSSG ratio can lead to excessive oxidative stress and inhibition of caspase-3-like activity, inducing cell death by necrosis, and, ultimately, resulting in the death of the organism. We therefore propose a reconsideration of the individual effective dose or individual tolerance concept postulated by Gaddum 50 years ago for the log-normal dose-response relationship. In addition, as NAC increased the tolerance to dichlorvos, it could be a potential antidote for OP poisoning, complementary to current treatments.
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PMID:Fish tolerance to organophosphate-induced oxidative stress is dependent on the glutathione metabolism and enhanced by N-acetylcysteine. 1456 51

Cruciferous vegetables contain glucosinolates that, after conversion to isothiocyanates (ITC), are capable of inducing cytoprotective genes. We examined whether broccoli seeds can elicit a chemoprotective response in mouse organs and rodent cell lines and investigated whether this response requires nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). The seeds studied contained glucosinolate at 40 mmol/kg, of which 59% comprised glucoiberin, 19% sinigrin, 8% glucoraphanin, and 7% progoitrin. Dietary administration of broccoli seeds to nrf2(+/+) and nrf2(-/-) mice produced a approximately 1.5-fold increase in NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) activities in stomach, small intestine, and liver of wild-type mice but not in mutant mice; increased transferase activity was associated with elevated levels of GSTA1/2, GSTA3, and GSTM1/2 subunits. These seeds also increased significantly the level of glutamate cysteine ligase catalytic (GCLC) subunit in the stomach and the small intestine of nrf2(+/+) mice but not nrf2(-/-) mice. An aqueous broccoli seed extract was prepared for treatment of cultured cells that contained ITC at approximately 600 mumol/L, composed of 61% 3-methylsulfinylpropyl ITC, 30% sulforaphane, 4% allyl ITC, and 4% 3-butenyl ITC. This extract induced GSTA1/2, GSTA3, NQO1, and GCLC between 3-fold and 10-fold in mouse Hepa-1c1c7 and rat liver RL-34 cells. The broccoli seed extract affected increases in GSTA3, GSTM1, and NQO1 proteins in nrf2(+/+) mouse embryonic fibroblasts but not in nrf2(-/-) mouse embryonic fibroblasts. These experiments show that broccoli seeds are effective at inducing antioxidant and detoxication proteins, both in vivo and ex vivo, in an Nrf2-dependent manner.
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PMID:Transcription factor Nrf2 is essential for induction of NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate cysteine ligase by broccoli seeds and isothiocyanates. 1557 60

The tripeptide glutathione (GSH) represents the major brain thiol and is essential for prevention of oxidative stress. Using monochlorobimane to label intracellular GSH in a glutathione S-transferase catalyzed reaction we have examined the kinetics of GSH metabolism including its rate of conjugation, total GSH content, synthesis, and efflux in astrocyte cultures under basal conditions and after induction of antioxidant response element (ARE)-mediated gene expression by the transcription factor Nrf2. In the presence of a cerebral spinal fluid-like salt solution astrocytes could not synthesize detectable levels of GSH. Addition of GSH precursors, cystine, glutamate, and glycine, rapidly restored GSH synthesis. Astrocytes were able to use either glutamate or glutamine as precursors equally for GSH synthesis. Using the small molecule chemical inducer tert-butylhydroqunione (tBHQ) we report that induction of ARE-mediated gene expression is associated with a coordinated increase in GSH content and synthesis rate with little effect on the rate of GSH conjugation or efflux. Consistent with the effect of the inducer, adenovirus-mediated overexpression of the transcription factor Nrf2 that mediates tBHQ's effects also increased GSH content, confirming that GSH metabolism can be regulated by the Nrf2 pathway.
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PMID:Coordinate regulation of glutathione metabolism in astrocytes by Nrf2. 1558 88

The chemopreventive effect of ethanol extract of Indigofera aspalathoides (EIA) on N-nitrosodiethylamine (DEN, 200 mg/kg)-induced experimental liver tumor was investigated in male Wistar rats. Oral administration of ethanol extract of Indigofera aspalathoides (250 mg/kg) effectively suppressed liver tumor induced with DEN as revealed by decrease in the levels of extend of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), total bilirubin, gamma glutamate transpeptidase (GGTP), lipid peroxidase (LPO), glutathione peroxidase (Gpx) and glutathione S-transferase (GST) with a concomitant increase in enzymatic antioxidant (superoxide dismutase and catalase) levels when compared to those in liver tumor bearing rats. The histopathological changes of liver sample were compared with respective control. Our results show a significant chemopreventive effect of EIA against DEN induced liver tumor.
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PMID:Chemoprevention of N-nitrosodiethylamine induced phenobarbitol promoted liver tumors in rat by extract of Indigofera aspalathoides. 1568 1

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.
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PMID:Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase. 1568 75


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