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)

CD4+ T cells specific for human cytomegalovirus (HCMV) IE1 protein are potential effectors of the control of HCMV infection through cytokine production. Better knowledge of major histocompatibility complex (MHC)-peptide-T cell receptor (TcR) interactions in the CD4+ T cell response should result in a better design of immunizing peptides and is a prerequisite for the development of vaccines or anti-cytomegalovirus therapy. In this study, the recombinant protein comprising residues 86-491 encoded by exon 4 of IE1 (GST-e4) was cleaved by enzymatic digestion and analyzed by high pressure liquid chromatography-mass spectroscopy (HPLC-MS). We identified the 14-residue epitope 162-DKREMWMACIKELH-175 recognized by an HLA-DR8-restricted clone, BeA3. Synthetic elongated, truncated and di-Ala-substituted peptides of the 18-mer IE1 158-IVPEDKREMWMACIKELH-175 sequence were used to analyze the amino acid motifs involved in binding to HLA-DR8 and recognition by the BeA3 clone. Substitutions which abolished (MW --> AA), or decreased (RE --> AA and MA --> AA) T cell clone proliferation, cytokine production and cytotoxicity were identified. Loss of T cell function induced by the MW --> AA substitution was associated with poor HLA-DR8 binding. Decreased T cell function (RE --> AA and MA --> AA) was associated with good HLA-DR8 binding, which suggested that these motifs were involved in TcR binding. Other substitutions induced potentiation of the T cell clone response: the IV --> AA substitution induced stronger proliferation, but equivalent cytokine production, when compared with the reference peptide IE1 (158-175). CI --> AA substitution induced strong potentiation of HLA-DR8 binding, proliferation and interferon-gamma and interleukin-4 production, possibly due to the removal of negative effects of Cys, Ile, or both side chains. Cytotoxicity was not improved by any substitution. Our results show modulation of the CD4+ T cell response according to the peptide residues involved in the HLA-DR8-peptide-TcR interaction.
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PMID:Characterization of an epitope of the human cytomegalovirus protein IE1 recognized by a CD4+ T cell clone. 864 75

Interleukin 1 (IL-1) potently activates human glomerular mesangial cells (HMC). In cytosolic extracts of IL-1-stimulated HMC or in anion exchange chromatography fractions we could not find any change in phosphorylation of myelin basic protein (MBP), a good substrate for extracellular regulated kinase (ERK). In contrast, IL-1 stimulated GST-jun kinase activity at least 10-fold. The jun kinase activity could be characterised as JNK1 and JNK2 at the protein and mRNA level. IL-1, TNF, UV light and osmotic stress, but not PMA, LPS, IL-3, IL-4, IL-6, IL-8, IL-10, IL-13, GM-CSF, PDGF, bFGF, TGF-beta and interferon-gamma were able to stimulate jun kinase activity in HMC, suggesting that jun kinase is selectively mediating signal transduction of the proinflammatory cytokines IL-1 and TNF as well as of cellular stress in HMC.
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PMID:Interleukin 1 activates jun N-terminal kinases JNK1 and JNK2 but not extracellular regulated MAP kinase (ERK) in human glomerular mesangial cells. 883 Jun 57

FIP-fve, a fungal immunomodulatory protein, was isolated from the fruiting bodies of the edible mushroom, Flammulina velutipes. FIP-fve was shown to stimulate blast-forming activity of human peripheral blood lymphocytes and gene expression of interleukin-2, interferon-gamma and tumor necrosis factor-alpha. Repeated administration of FIP-fve to mice inhibits the Arthur and systemic anaphylaxis reactions. FIP-fve cDNA was cloned and sequenced, and the amino acid sequence of FIP-fve deduced from the nucleotide sequence is identical to that previously determined by protein sequencing. FIP-fve cDNA was amplified by polymerase chain reaction, ligated into the expression vector, pGEX-2T, and expressed in Escherichia coli as a fusion protein of glutathione S-transferase (GST) and FIP-fve. The GST-FIP-fve fusion protein was soluble, and the yield of recombinant FIP-fve was about 5 mg/L of induced culture. The recombinant FIP-fve was obtained by cleaving the GST-FIP-fve fusion protein with thrombin and purifing to homogeneity. The recombinant FIP-fve had about 50% of the immunomodulatory activity of the native FIP-fve.
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PMID:Molecular cloning and expression of a fungal immunomodulatory protein, FIP-fve, from Flammulina velutipes. 926 56

We recently identified SH2-Bbeta as a JAK2-binding protein and substrate involved in the signaling of receptors for growth hormone and interferon-gamma. In this work, we report that SH2-Bbeta also functions as a signaling molecule for platelet-derived growth factor (PDGF). SH2-Bbeta fused to glutathione S-transferase (GST) bound PDGF receptor (PDGFR) from PDGF-treated but not control cells. GST fusion protein containing only the SH2 domain of SH2-Bbeta also bound PDGFR from PDGF-treated cells. An Arg to Glu mutation within the FLVRQS motif in the SH2 domain of SH2-Bbeta inhibited GST-SH2-Bbeta binding to tyrosyl-phosphorylated PDGFR. The N-terminal truncated SH2-Bbeta containing the entire SH2 domain interacted directly with tyrosyl-phosphorylated PDGFR from PDGF-treated cells but not unphosphorylated PDGFR from control cells in a Far Western assay. These results suggest that the SH2 domain of SH2-Bbeta is necessary and sufficient to mediate the interaction between SH2-Bbeta and PDGFR. PDGF stimulated coimmunoprecipitation of endogenous SH2-Bbeta with endogenous PDGFR in both 3T3-F442A and NIH3T3 cells. PDGF stimulated the rapid and transient phosphorylation of SH2-Bbeta on tyrosines and most likely on serines and/or threonines. Similarly, epidermal growth factor stimulated the phosphorylation of SH2-Bbeta; however, phosphorylation appears to be predominantly on serines and/or threonines. In response to PDGF, SH2-Bbeta associated with multiple tyrosyl-phosphorylated proteins, at least one of which (designated p84) does not bind to PDGFR. Taken together, these data strongly argue that, in response to PDGF, SH2-Bbeta directly interacts with PDGFR and is phosphorylated on tyrosine and most likely on serines and/or threonines, and acts as a signaling protein for PDGFR.
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PMID:Platelet-derived growth factor (PDGF) stimulates the association of SH2-Bbeta with PDGF receptor and phosphorylation of SH2-Bbeta. 969 82

Fc receptors modulate inflammatory processes, including phagocytosis, serotonin and histamine release, superoxide production, and secretion of cytokines. Aggregation of FcgammaRIIa, the low-affinity receptor for monomeric IgG, activates nonreceptor protein tyrosine kinases such as Lyn, Hck, and Syk, potentially driving the phosphorylation of the downstream adaptor proteins, including Cbl and/or Nck. Previous work from our laboratory using interferon-gamma-differentiated U937 (U937IF) myeloid cells investigated mechanisms which regulate Fcgamma receptor-induced assembly of adaptor complexes. Herein we report that FcgammaRII receptor signaling in U937IF and HEL cells involves Cbl and Nck, suggesting that Cbl-Nck interactions may link FcgammaRII to downstream activation of Pak kinase. FcgammaRII crosslinking induced the phosphorylation of Cbl and Nck on tyrosine. The alphaCbl immunoprecipitations revealed constitutive binding of Nck and Grb2 to Cbl and FcgammaRII-inducible binding of CrkL to Cbl. The interactions of Cbl with Nck and CrkL were phosphorylation dependent since dephosphorylation of cellular proteins with potato acid phosphatase abrogated binding. GST-Nck fusion protein pulldown experiments show that Cbl and Pak1 bind to the second SH3 domain of Nck. A specific Src inhibitor, PP1, was shown to completely abrogate the FcgammaR-induced superoxide response, correlating with a decrease in Cbl and Nck tyrosine phosphorylation. Our results provide the first evidence that Src is required for FcgammaR activation of the respiratory burst in myeloid cells and suggest that Cbl-Nck, Cbl-Pak1, and Nck-Pak1 interactions may regulate this response.
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PMID:Characterization of Cbl-Nck and Nck-Pak1 interactions in myeloid FcgammaRII signaling. 985 74

Food contaminants may contribute to the recent increased incidence of food allergies. We have investigated this hypothesis experimentally. It was our objective to determine whether toxicity to the intestinal tissue by orally applied mercury (Hg) could modulate the immune response to food allergens. Effective mechanisms were studied with functional immunological and toxicological parameters. Brown Norway rats were immunized intraperitoneally by ovalbumin (OVA). Before oral challenge with OVA, immunized and non-immunized animals were exposed to HgCl2. Immunological responses were measured by enzyme-linked immunosorbent assays [anti-OVA-IgE and-IgG, rat mast cell protease II (RMCPII), interferon-gamma, interleukin-4, lymphocyte proliferation] and by flow cytometry (lymphocyte subpopulations). Toxicity of Hg to the intestinal barrier was determined by measuring viability, DNA damage and induction of glutathione S-transferase in isolated intestinal epithelial cells and lymph node cells, and by measuring permeability, short-circuit current and tissue conductance of the intact intestinal epithelium. A single high oral dose of HgCl2 enhanced the serum concentrations of anti-OVA-IgE and IgG (P < 0.05) and of RMCPII (P < 0.05) in immunized rats. The treatment resulted in a higher number of CD4/CD25+ T cells in the lymph nodes (P < 0.05). The multiple application of low HgCl2 doses (5 x 0.2 mg/kg body weight) only resulted in an elevated RMCPII serum concentration (P < 0.05). Neither treatment schedules impaired proliferation and cytokine production of lymphocytes. In non-immunized rats only minor immunological changes were observed. Oral HgCl2 induced genotoxic damage in lymph node cells and in jejunal epithelial cells (P < 0.05). Moreover, HgCl2 increased the permeability of intestinal epithelial tissue and of Caco-2 monolayers and was genotoxic and cytotoxic to isolated intestinal epithelial cells in vitro. In conclusion, these studies indicate that the food contaminant Hg can stimulate the immune response to OVA in immunized rats. One possible mechanism could be the toxicity of Hg to the intestinal epithelial and the lymph node cells. Whether humans with allergies respond to high oral doses of Hg in a similar way needs to be investigated in further studies.
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PMID:Enhancement of ovalbumin-induced antibody production and mucosal mast cell response by mercury. 1047 31

At present, only a little is known about the transcriptional regulation in chondrocytes submitted to various physicomechanical factors known to exist in articular cartilage. Recently, we have investigated the effects of hydrostatic pressure on transcriptional control in chondrocytes using human chondrosarcoma and immortalized chondrocyte cell lines for the experiments. Hydrostatic pressure was applied on the cells in a special computer-controlled, water-filled pressure chamber, where cyclic and static pressures up to 32 MPa can be created. Differential display RT-PCR and probing of cDNA arrays are the methods we have used to study differential gene expression due to hydrostatic pressure. By differential display RT-PCR experiments, we have observed several differentially expressed cDNA bands under continuous 30 MPa hydrostatic pressure, while 30 MPa cyclic pressure at 1 Hz produced much fewer changes. In the first phase of our studies, we have focused on the effects of 30 MPa hydrostatic pressure because it causes a unique hsp70-mediated stress response in immortalized chondrocytes. Differential display RT-PCR screening provided us with several clones that derive from low-abundance mRNAs, such as death-associated protein 3 (DAP3), a nucleotide-binding protein which increases due to interferon-gamma induced cell death; PTZ-17 (or p311), a seizure-related protein; H-NUC, a nuclear DNA binding protein; and one new gene of unknown function. In Northern blots, an induction was confirmed for the new gene, DAP3 and PTZ-17 were down-regulated in some but not in all parallel experiments; however, basal level of H-NUC mRNA was too low to be detected in Northern blots. We then chose to widen our screening to a number of known genes arrayed as cDNA blots. Under 30 MPa continuous hydrostatic pressure, four different time points were chosen (0, 3, 6 and 24 h) for the experiments. The screening of 588 cDNAs showed 15 up-regulated and 6 down-regulated genes. Consistently with our previous results hsp70 was highly induced, as well as hsp40, a chaperone protein functioning together with hsp70. Gadd45 and to a lesser extent Gadd153 (stress genes induced by, e.g., ionizing radiation and ischaemia) were up-regulated, as well as p21waf1,cip1, a protein participating in cell cycle regulation that can interact with Gadd45. Northern blots confirmed Gadd45 induction. Down-regulated transcripts included, e.g., DAD-1, glutathione S-transferase pI, DNA-binding inhibitor ID-1H, and cytoplasmic dynein light chain.
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PMID:Transcriptional activation in chondrocytes submitted to hydrostatic pressure. 1091 81

We generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSSLLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p43) and a C-terminal one (p12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD(376)GPAMKNVEF, identified on N-terminal sequencing of the p12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblotting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-gamma-treated U937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U937 cells treated with vitamin D(3) or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U937 differentiation occur upstream of caspase-8 processing/activation.
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PMID:Monitoring of caspase-8/FLICE processing and activation upon Fas stimulation with novel antibodies directed against a cleavage site for caspase-8 and its substrate, FLICE-like inhibitory protein (FLIP). 1209 60

The inducible nitric oxide synthase (iNOS) gene plays an important role in renal diseases. Transcription is the principal mode of regulation. This study explores the role of acetylation in cytokine-mediated iNOS induction in cultured murine mesangial cells and RAW 264.7 cells. Nitric oxide production was measured by the Griess reaction. The activity of the iNOS promoter and a nuclear factor-kappa B (NF-kappa B) element promoter were assessed in transient transfection assays. Gel shift and supershift assays were used to identify NF-kappa B in nuclear extracts. Protein-protein interactions were assayed by co-immunoprecipitation and GST pull-down assays. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and overexpression of HDAC isoforms were used to assess the impact of acetylation status on iNOS and NF-kappa B element promoter activity. TSA inhibited induction of endogenous NO production and iNOS as well as NF-kappa B element promoter activity in response to interleukin-1 beta (IL-1 beta) or lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) in both cell types without altering NF-kappa B DNA binding activity. Overexpression of specific HDAC isoforms enhanced cytokine induction of both the iNOS and the NF-kappa B element promoter. HDAC2 and NF-kappa B p65 co-immunoprecipitated from mesangial cell nuclear extracts, and in vitro translated HDAC2 specifically interacted with an NF-kappa B p65 GST fusion protein. Hyperacetylation diminishes cytokine induction of iNOS transcription activity, at least partially, by limiting the functional efficacy of NF-kappa B. The specific recruitment of HDAC2 to NF-kappa B at target promoters and the consequent effects on acetylation status may play an important role in regulating iNOS as well as other NF-kappa B-dependent genes involved in inflammation.
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PMID:Histone deacetylases augment cytokine induction of the iNOS gene. 1213 31

Signal transducer and activator of transcription (STAT) proteins are both tyrosine- and serine-phosphorylated, mediating signal transduction and gene regulation. Following gene regulation, STAT activity in the nucleus is then terminated by a nuclear protein phosphatase(s), which remains unidentified. Using novel antibody arrays to screen the Stat1-specific protein phosphatase(s), we identified a SHP-2-Stat1 interaction in the A431 cell nucleus. SHP-2 and Stat1 nuclear localization and their association in response to either epidermal growth factor or interferon-gamma (IFNgamma) were confirmed by immunofluorescent staining and affinity precipitation assays. The SHP-2 C-terminal region containing protein-tyrosine phosphatase activity interacted with the C-terminal SH2 transcriptional activation domain of Stat1. In SHP-2-/- mouse fibroblast cells, Stat1 phosphorylation at both the tyrosine residue Tyr(701) and the serine residue Ser(727) by IFNgamma was enhanced and prolonged. Consistently, purified GST-SHP-2 dephosphorylated Stat1 at both tyrosine and serine residues when immunoprecipitated phospho-Stat1 or a peptide corresponding to the sequence surrounding Tyr(P)(701) or Ser(P)(727) of Stat1 was used as the substrate. Overexpression of SHP-2 in 293T cells inhibited IFNgamma-dependent Stat1 phosphorylation and suppressed Stat1-dependent induction of luciferase activity. Our findings demonstrate that SHP-2 is a dual-specificity protein phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues and plays an important role in modulating STAT function in gene regulation.
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PMID:SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. 1227 Sep 32


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