Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.
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PMID:Combined effects of gamma radiation and arsenite on the proteome of human TK6 lymphoblastoid cells. 1572 13

In the current study, the toxicological mechanisms of microcystin-LR and its disadvantageous effects on Daphnia magna were examined. Survival rate, number of newborn, activity of several important enzymes [glutathione S-transferase (GST), lactate dehydrogenase (LDH), phosphatases, and glutathione], accumulated microcystins, and ultrastructural changes in different organs of Daphnia were monitored over the course of 21-day chronic tests. The results indicated that low concentrations of dissolved microcystin had no harmful effect on Daphnia. On the contrary, stimulatory effects were detected. In the presence of toxin at high dosage and for long-term exposure, GST and glutathione levels decreased significantly. The decreased enzyme activity in the antioxidant system probably was caused by detoxification reactions with toxins. And these processes of detoxification at the beginning of chronic tests may enable phosphatases in Daphnia magna to withstand inhibition by the toxins. At the same time, we also found that the LDH activity in test animals increased with exposure to microcystin-LR, indicating that adverse effects occurred in Daphnia. With microcystin given at a higher dosage or for a longer exposure, the effect on Daphnia magna was fatal. In the meantime, microcystin began to accumulate in Daphnia magna, and phosphatase activity started to be inhibited. From the ultrastructure results of cells in D. magna, we obtained new information: the alimentary canal may be the target organ affected by exposure of microcystins to D. magna. The results of the current study also suggested that the oxidative damage and PPI (protein phosphatase inhibition) mechanisms of vertebrates also are adapted to Daphnia.
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PMID:Chronic toxicity and responses of several important enzymes in Daphnia magna on exposure to sublethal microcystin-LR. 1589 60

Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor.
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PMID:NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin. 1600 30

Activation of NFAT transcription factors requires their dephosphorylation by the phosphatase calcineurin (CN). NFATs contain two CN binding motifs: PxIxIT and CnBP-B/CNBR2 (which we call LxVP). Here we carry out a detailed comparative analysis of the CN binding activity displayed by the PxIxIT and LxVP sites from different NFATs. Dose-response CN binding experiments with GST fusion proteins of NFATc1 and NFATc2 showed that NFATc1 binds CN in vitro more efficiently than does NFATc2. This difference in binding appears to be caused by the different CN binding potencies of the corresponding LxVP sites; thus while the LxVPc2 peptide fused to GST did not bind CN, GST-LxVPc1 bound it more efficiently than did GST-PxIxITc1 or GST-PxIxITc2. Furthermore, an NFATc2 chimera protein containing the LxVP motif from NFATc1 interacted with CN much more potently than did wild-type NFATc2. Free peptides spanning the LxVP motifs from NFATc1, c3 or c4 displaced CN from GST-NFATc1 and GST-NFATc2 more efficiently than any PxIxIT peptide. PxIxITc2 and LxVPc1 peptides were each able to cross-compete GST-LxVPc1-CN and GST-PxIxITc2-CN binding. In contrast with PxIxITc2, the LxVP peptide not only blocked CN-NFAT binding but also inhibited CN phosphatase activity in vitro. Furthermore, exogenous LxVPc1 blocked NFATc2 phosphorylation and nuclear translocation in vivo. These results suggest a model in which the different CN binding characteristics of the PxIxIT and LxVP sites enable different NFAT members to influence each others activities in cells where they are co-expressed.
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PMID:Blockade of NFAT activation by the second calcineurin binding site. 1640 84

The suppressor of the dis2 mutant (sds22+) has been shown to be an essential regulator in cell division of fission and budding yeast where its deletion causes mitotic arrest. Its role seems to take place through the activation of PP1 (protein phosphatase type 1) in Schizosaccharomyces pombe. In the trematode Schistosoma mansoni, we have identified the Sds22 homologue (SmSds), and the PP1 (SmPP1). We showed by using a GST (glutathione S-transferase) pull-down assay that the SmSds gene product interacts with SmPP1 and that the SmSds-SmPP1 complex is present in parasite extracts. Furthermore, we observed that SmSds inhibited PP1 activity. Functional studies showed that the microinjection of SmSds into Xenopus oocytes interacted with the Xenopus PP1 and disrupted the G2/M cell-cycle checkpoint by promoting progression to GVBD (germinal vesicle breakdown). Similar results showing the appearance of GVBD were observed when oocytes were treated with anti-PP1 antibodies. Taken together, these observations suggest that SmSds can regulate the cell cycle by binding to PP1.
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PMID:Characterization of Schistosoma mansoni Sds homologue, a leucine-rich repeat protein that interacts with protein phosphatase type 1 and interrupts a G2/M cell-cycle checkpoint. 1641 88

Down syndrome critical region gene 1-like 2 (DSCR1L2) belongs to the human DSCR1-like gene family, which also includes DSCR1 and DSCR1L1. Both DSCR1 and DSCR1L1 proteins interact with calcineurin, a calcium/calmodulin-dependent phosphatase. To date, no interactor has been described for DSCR1L2. The aim of this work was to perform a first functional study of DSCR1L2 using yeast two-hybrid analysis conducted on a human heart cDNA library. Here, we report the interaction between DSCR1L2 and the human cardiac troponin I (TNNI3), the heart-specific inhibitory subunit of the troponin complex, a central component of the contractile apparatus. This interaction was confirmed by both yeast cotransformation and GST (glutathione-sepharose transferase) fusion protein assay. Moreover, a new DSCR1L2 mRNA isoform, generated by alternative splicing, was identified and cloned in different tissues: it lacks two central exons, encoding the most conserved domains among the DSCR1-like protein family. A quantitative relative reverse transcription-polymerase chain reaction (RT-PCR) assay showed that in heart tissue the normalized expression level ratio for DSCR1L2 and DSCR1L2-E2E5 mRNA isoforms is 3.5:1, respectively. The yeast cotransformation and GST fusion protein assay demonstrated the interaction between this new DSCR1L2 variant and the human cardiac troponin I and the prominent role of DSCR1L2 exon 2 in determining binding between both DSCR1L2 isoforms and TNNI3. These data indicate an entirely new role for a DSCR1-like family gene, suggesting a possible involvement of DSCR1L2 in cardiac contraction.
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PMID:Proteins encoded by human Down syndrome critical region gene 1-like 2 (DSCR1L2) mRNA and by a novel DSCR1L2 mRNA isoform interact with cardiac troponin I (TNNI3). 1651 8

The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel) is activated in response to the calcium signal by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study we report that calcineurin also interacts with TRESK via an NFAT-like docking site, in addition to its enzymatic action. In its intracellular loop, mouse TRESK possesses the amino acid sequence, PQIVID, which is similar to the calcineurin binding consensus motif, PXIXIT (where X denotes any amino acids), necessary for NFAT (nuclear factor of activated T cells) activation and nuclear translocation. Mutations of the PQIVID sequence of TRESK to PQIVIA, PQIVAD, or PQAVAD increasingly deteriorated the calcium-dependent activation in the listed order and correspondingly reduced the benzocaine sensitivity (a property discriminating activated channels from resting ones), when it was measured after the calcium signal in Xenopus oocytes. Microinjection of VIVIT peptide, designed to inhibit the NFAT-calcineurin interaction specifically, also eliminated TRESK activation. The intracellular loop of TRESK, expressed as a GST fusion protein, bound constitutively active calcineurin in vitro. PQAVAD mutation as well as addition of VIVIT peptide to the reaction abrogated this calcineurin binding. Wild type calcineurin was recruited to GST-TRESK-loop in the presence of calcium and calmodulin. These results indicate that the PQIVID sequence is a docking site for calcineurin, and its occupancy is required for the calcium-dependent regulation of TRESK. Immunosuppressive compounds, developed to target the NFAT binding site of calcineurin, are also expected to interfere with TRESK regulation, in addition to their desired effect on NFAT.
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PMID:Targeting of calcineurin to an NFAT-like docking site is required for the calcium-dependent activation of the background K+ channel, TRESK. 1656 37

The virulence of the opportunistic pathogen Pseudomonas aeruginosa (Pa) is in part mediated by the type III secretion (TTS) of bacterial proteins into eukaryotic hosts. Exoenzyme S (ExoS) is a bifunctional Pa TTS effector protein, with GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities. Known cellular substrates of TTS-translocated ExoS (TTS-ExoS) ADPRT activity include proteins in the Ras superfamily and ERM family proteins. This study describes the ADP-ribosylation of a non-G-protein substrate of TTS-ExoS, cyclophilin A (CpA), a peptidyl-prolyl isomerase (PPIase). Four novel 17 kDa proteins (pI 6.5-6.8) were recognized in a proteomic screen of lysates of human epithelial cells that had been exposed to ExoS-producing Pa, but not an isogenic non-ExoS producing strain. The proteins were identified as isoforms of CpA using MALDI-TOF mass spectrometry and confirmed by Western blotting. Mutagenesis analysis identified arginine 55 and 69 of CpA as sites of ExoS ADP-ribosylation. Examination of the effect of ExoS ADP-ribosylation on CpA function found a moderate (19%) decrease in prolyl isomerization of a Xaa-Pro containing peptides. In comparison, GST-CpA co-immunoprecipitation studies found ExoS ADP-ribosylation of CpA to efficiently inhibit CpA binding to calcineurin/PP2B phosphatase. Our results support that ExoS ADP-ribosylates and affects the function of the cytosolic protein, CpA, with the predominant functional effect relating to interference of CpA-cellular protein interactions.
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PMID:ADP-ribosylation of cyclophilin A by Pseudomonas aeruginosa exoenzyme S. 1658 1

G(q) protein-coupled receptor stimulation increases sarcolemmal Na(+)/H(+) exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A(1) receptor stimulation inhibits this response through a G(i) protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A(1) receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the alpha(1)-adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A(c)) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G(i) proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A(c) and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A(c) dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A(1) receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G(i) protein-mediated translocation of PP2A(c) and NHE1 dephosphorylation.
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PMID:A novel role for protein phosphatase 2A in receptor-mediated regulation of the cardiac sarcolemmal Na+/H+ exchanger NHE1. 1670 1

Lafora disease is a progressive myoclonus epilepsy with an early fatal issue. Two genes were identified thus far, the mutations of which cause the disease. The first one, EPM2A, encodes the consensus sequence of a protein tyrosine phosphatase. Its product, laforin, is the object of the present work. We analysed in detail the amino acid sequence of this protein. This suggested, as also observed by others, that it could present two domains, a carbohydrate-binding domain (CBM20, known as a starch-binding domain) and the catalytic domain of a dual-specificity protein phosphatase. We produced the enzyme as two different GST-fused proteins and as an N-terminally His-tagged protein. Differences in solubility were observed between the constructs. Moreover, the N-terminal carbohydrate-binding domain contains a thrombin cleavage site, which is hidden in the simplest GST-fusion protein we produced, but was accessible after introducing a five-residue linker between the engineered cleavage site and the enzyme N-terminus. The two types of constructs hydrolyse pNPP and OMFP with kinetic parameters consistent with those of a dual-specificity phosphatase. We show in addition that the protein not only binds glycogen, but also starch, amylose and cyclodextrin. Neither binding of glycogen nor of beta-cyclodextrin appreciably affects the phosphatase activity. These results suggest that the role of the N-terminal domain is rather that of targeting the protein in the cell, probably to glycogen and the protein complexes attached to it, rather than that of directly modulating the catalytic activity.
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PMID:Molecular characterization of laforin, a dual-specificity protein phosphatase implicated in Lafora disease. 1701 Apr 95


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