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)

Signal transduction by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin) is mediated by the intracellular dioxin receptor which, in its dioxin-activated state, regulates transcription of target genes encoding drug metabolizing enzymes such as cytochrome P-450IA1 and glutathione S-transferase Ya. Upon binding of dioxin the receptor translocates from the cytoplasm to the nucleus in vivo and is converted from a latent non-DNA binding form to a species which binds to dioxin-responsive positive control elements in vitro. The latent receptor form is associated with an inhibitory protein (the 90-kDa heat shock protein, hsp90), the release of which is necessary to unmask the DNA binding activity of the receptor. Here we have established a protocol to disrupt the hsp90-receptor complex in the absence of ligand. We show that it was possible to covalently cross-link with dioxin only the hsp90-associated form of dioxin receptor. In contrast, the disrupted hsp90-free form of receptor did not form a stable complex with dioxin but bound DNA constitutively. Moreover, we could partially reconstitute the ligand binding activity of the salt-disrupted hsp90-free dioxin receptor by incubation with hsp90-containing reticulocyte lysate but not by incubation with wheat germ lysate which lacks immuno-detectable levels of hsp90. Thus, we demonstrate that the dioxin receptor loses its high affinity ligand binding activity following release of hsp90 and that it is possible to reverse this process. In conclusion, hsp90 appears to play dual roles in the modulation of functional activities of the dioxin receptor: (i) it represses the intrinsic DNA binding activity of the receptor and (ii) it appears to determine the ability of the receptor to assume and/or maintain a ligand binding conformation.
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PMID:Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. 132 28

We expressed the carboxyl-terminal 178 amino acids of the rabbit cardiac Na+/H+ exchanger as a fusion protein with glutathione-S-transferase. The fusion protein (PCR178) was found in the supernatant of extracts of E. coli and was purified using Glutathione-Sepharose affinity chromatography. Affinity-purified antibodies raised against the carboxyl-terminal region of the Na+/H+ exchanger identified the resultant protein. PCR178 copurified with a 70 kDa protein. Amino-terminal sequencing of the 70 kDa protein identified it as dnaK, the bacterial equivalent of the mammalian 70 kDa heat shock protein (hsp70). DnaK was dissociated from the Na+/H+ exchanger fusion protein by the addition of MgATP. When purified PCR178 was coupled to a cyanogen bromide-activated Sepharose column, bovine hsp70 bound to the column and was eluted with MgATP. Nondenaturing polyacrylamide gel electrophoresis showed that, in the absence of MgATP, hsp70 formed a complex with PCR178. The complex was dissociated by the addition of MgATP. GST alone did not form a complex with hsp70. Immunoprecipitation of the Na+/H+ exchanger with antiexchanger antibodies resulted in coprecipitation of hsp70 protein from antiporter containing cells. Cells that overexpress the Na+/H+ exchanger had increased amounts of hsp70 which coprecipitated with antiexchanger antibody. The results show that heat shock protein complexes with the mammalian Na+/H+ exchanger.
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PMID:The carboxyl-terminal region of the Na+/H+ exchanger interacts with mammalian heat shock protein. 765 95

The major water-soluble proteins--or crystallins--of the eye lens are either identical to or derived from proteins with non-refractive functions in numerous tissues. In general, the recruitment of crystallins has come from metabolic enzymes (usually with detoxification functions) or stress proteins. Some crystallins have been recruited without duplication of the original gene (i.e., lactate dehydrogenase B and alpha-enolase), while others have incurred one (i.e., argininosuccinate lyase and a small heat shock protein) or several (i.e., glutathione S-transferase) gene duplications. Enzyme (or stress protein)-crystallins often maintain their non-refractive function in the lens and/or other tissues as well as their refractive role, a process we call gene sharing. alpha-Crystallin/small heat shock protein/molecular chaperone is of special interest since it is the major crystallin of humans. There are two alpha-crystallin genes (alpha A and alpha B), with alpha B retaining the full functions of a small heat shock protein. Here we describe recent evidence indicating that alpha A and alpha B have kinase activity, which would make them members of the enzyme-crystallins. We also describe various regulatory elements of the mouse alpha-crystallin genes responsible for their expression in the lens and, for alpha B, in skeletal muscle. Delineating the control elements for gene expression of these multifunctional protective proteins provides the foundations for their eventual use in gene therapy. Finally, comparison of the mouse and chicken alpha A-crystallin genes reveals similarities and differences in their functional cis-acting elements, indicative of evolution at the level of gene regulation.
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PMID:Recruitment of enzymes and stress proteins as lens crystallins. 803 55

The isolation, characterization, and expression of a novel cDNA encoding a Trypanosoma cruzi polypeptide (TcAc2), homologous to various small stress proteins and glutathione S-transferases, are described. The deduced amino-acid sequence revealed two domains sharing 27% identity and an additional 27% similarity to each other suggesting that the molecule may have evolved from a single domain by a process of gene duplication and fusion. The TcAc2 cDNA was subcloned into the pGEX-2T vector for expression in E coli. In vitro translation products of epimastigote mRNA, immunoprecipitated with anti-TXepi serum, showed a major radioactive band of 52 kDa. Immunoprecipitation of [35S]methionine labelled epimastigote and trypomastigote antigens after pulse chase experiments, using anti-TcAc2 fusion protein antibodies, showed that the protein is released into the culture medium. Moreover, Western blot analysis revealed a single band of 52 kDa with epimastigote, trypomastigote and amastigote antigens. Primary structure homology searches revealed that each TcAc2 domain contained within its N-terminus significant homology to Solanum tuberosum pathogenesis-related protein PR1, soybean heat shock protein 26-A, auxin regulated clone pCNT103 from Nicotiana tabacum and Drosophila melanogaster glutathione S-transferase 27 (GST27). This finding was supported by a comparison of hydrophobicity profiles of TcAc2 and these proteins. Most of them play a central role in protection mechanisms against stress. Based on the homology between TcAc2, glutathione S-transferases (GST) and small stress proteins, it is likely that the TcAc2 gene product may play a crucial role in parasite's adaptation to its microenvironment. These molecules could be considered as members of the GST superfamily, where the T cruzi protein may take a particular place because of its internal gene duplication.
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PMID:Trypanosoma cruzi cDNA encodes a tandemly repeated domain structure characteristic of small stress proteins and glutathione S-transferases. 805 80

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics. 839 10

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts, has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with 35[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3) within the pro alpha 1 (I)- N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a "chaperone" role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.
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PMID:Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain. 856 53

Domain E, considered as the putative hormone binding domain (HBD) of the human mineralocorticoid receptor (hMR) was expressed in Escherichia coli as a fusion protein with either maltose binding protein (MBP) or glutathione S-transferase (GST). These bacterially-produced MR constructs had no steroid binding activity per se. In fact, heat shock protein association (hsp) is required for high affinity ligand-binding of the MR. After incubation of purified MBP- or GST-HBD with rabbit reticulocyte lysate, known to be rich in heat shock proteins, we obtained saturable binding of [3H]aldosterone. The Kd value for aldosterone was 0.3 nM and the Bmax = 32 pmol/mg. Hormone binding specificity was assessed by competition studies with various steroid ligands. Sucrose gradient assays performed with [3H]aldosterone-MBP-HBD revealed complex sedimenting at 8.3S and 4.9S with [3H]progesterone-MBP-HBD. Western-blot analysis of the sedimentation peak showed the concomitant presence of MBP-HBD by a monoclonal anti-MBP antibody, and hsp90 by a monoclonal anti-hsp antibody. Moreover, following incubation with the anti-rabbit hsp90 monoclonal antibody the sedimenting gradient showed a 10.4S sedimenting complex. These analyses demonstrated that the [3H]aldosterone-MBP-HBD complex is at least associated with hsp90 in reticulocyte lysate and that the HBD of hMR is sufficient to bind hsp90. Deletions of a relatively short amino- (729-766) or carboxy-terminal (940-984) region of the HBD fragment eliminated all steroid-binding properties. Overall, these results indicate that the integrity of domain E is necessary and sufficient to bind steroid ligands, agonists or antagonists, with characteristics similar to the entire native MR.
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PMID:Putative steroid binding domain of the human mineralocorticoid receptor, expressed in E. coli in the presence of heat shock proteins shows typical native receptor characteristics. 864 16

Bacterial LPS stimulation of murine macrophages leads to increased tyrosine phosphorylation and activation of the 42- and 44-kDa mitogen-activated protein kinases (MAPK) and the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinase (JNK) and p38, related to the high osmolarity glycerol protein kinase in Saccharomyces cerevisiae (HOG1). LPS caused a rapid increase (10 min) in phosphotransferase activity toward myelin basic protein (MBP), a polypeptide that encompassed the first 169 residues of c-Jun fused to gluthathione S-transferase (GST-c-Jun (1-169)) and 27-kDa heat shock protein (hsp27). MonoQ fractionation of cell extracts resolved phosphotransferase activity peaks toward MBP, GST-c-Jun (1-169), and hsp27, which contained MAPK, SAPK/JNK, and MAPKAPK2, respectively, as indicated by immunoblotting data. In RAW 264.7 macrophages, LPS stimulation of MAPKAPK2, a substrate of p38 HOG1 and MAPK, appeared to occur predominantly via p38 HOG1 and not the MAPK. PMA, which activated the MAPK as potently as LPS, did not strongly activate MAPKAPK2, as assessed by hsp27 phosphorylation. Consistent with p38 HOG1-mediating LPS activation of MAPKAPK2, treatment with LPS, but not PMA, increased the tyrosine phosphorylation of p38 HOG1, a modification known to elevate the enzymatic capacity of this kinase. In LPS-treated cells, the activity of SAPK/JNK was increased 5- to 10-fold, as measured by precipitating SAPK/JNK with Abs or immobilized GST-c-Jun and performing an in vitro kinase assay. In addition, the kinases thought to be upstream of SAPK/JNK, SAPK/ERK kinase 1 (SEK1), and MAPK/ERK kinase kinase 1 (MEKK1), were activated following LPS, but not PMA, exposure (5-fold and 2.5-fold, respectively.
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PMID:Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages. 866 21

A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus. In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation. Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule. The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli.
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PMID:A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock. 875 26

In intact cells, mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 is rapidly activated by various cytokines, stresses, and chemotactic factors. The small heat shock protein p27 has been shown to be a substrate for MAPKAP kinase 2. Recently, we identified a novel substrate, designated p60, for MAPKAP kinase 2 in human neutrophils (Zu, Y.-L., Ai, Y., Gilchrist, A., Labadia, M. E., Sha'afi, R. I., and Huang, C.-K. (1996) Blood 87, 5287-5296). To further understand the signaling pathway of MAPKAP kinase 2, we have purified p60 from a heat-treated neutrophil lysate by DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis. Microsequencing of five peptides derived from purified p60 indicates that p60 is lymphocyte-specific protein 1 (LSP1). Furthermore antibodies specific for human and mouse LSP1 react with human and mouse p60. The sequence of human LSP1 indicates two serine residues at positions 204 and 252 as potential phosphorylation sites. The amino acid sequences surrounding these two sites are in agreement with the consensus sequence (Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa) for phosphorylation by MAPKAP kinase 2. Both serine residues in human LSP1 and the corresponding conserved serine residues in mouse LSP1 are in the basic C-terminal F-actin binding domain. Various fusion proteins of wild type and truncated mouse LSP1 with glutathione S-transferase were tested for their capacity to be phosphorylated by MAPKAP kinase 2. The results indicate that LSP1 is a substrate for MAPKAP kinase 2 in vitro and that the phosphorylation sites are located in the basic C-terminal domain of LSP1. Because both the small heat shock proteins and LSP1 are F-actin binding proteins, these results suggest a role for MAPKAP kinase 2 in the regulation of cytoskeletal structure or function.
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PMID:LSP1 is the major substrate for mitogen-activated protein kinase-activated protein kinase 2 in human neutrophils. 899 17


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