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)

High levels of expression of GSTP1-1 are associated with cell proliferation, embryogenesis and malignancy. Given the role of glutathione S-transferase (GST) in detoxication, it is possible that GSTP1-1 evolved specifically to protect proliferating cells and share regulatory mechanisms with other cellular genes which are involved in cell division and tumorigenesis. We have previously shown that the expression of GSTP1 is suppressed by retinoic acid (RA) in the presence of the retinoic acid receptor (RAR) as a result of decreased transcription from its promoter. Through deletion analysis, we show here that the RA-RAR-dependent repression is mediated by the region -73 to +8. Further mutation analysis of this region indicates that the DNA sequence required for RA-RAR-dependent repression co-localizes with a consensus activator protein-1 (AP1) site essential for the promoter activity. The degree of repression correlates with the residual activity of the AP1 site. There are two adjacent G/C boxes. The one immediately downstream from the AP1 site is not essential for the promoter activity, but mutation of the second, further downstream, impairs the promoter. On the other hand, mutation of either of these two G/C boxes has little effect on RA-RAR suppression. We also show that the expression of GSTP1 is regulated by the redox status of the cell. Using the chloramphenicol acetyltransferase assay system, we have demonstrated that treatment with H2O2 induced transcription from the promoter and that this effect can be blocked by pre-incubation with N-acetylcysteine (NAC). It was shown that the induction by H2O2 is mediated by trans-acting factor NF-kappa B (nuclear factor kappa B), via a putative NF-kappa B site, 'GGGACCCTCC', located from -96 to -86. Co-transfection with an NF-kappa B (p65) expression construct increased the promoter activity, an effect which could be blocked by co-transfection with an I kappa B (MAD-3) expression construct. Deletion of the NF-kappa B site abolished the effect of both H2O2 and co-transfection of NF-kappa B. Interestingly, NAC is also an inducer for GSTP1. The effect of NAC was shown to be mediated largely by the AP1 site, since mutation of this site abolished the induction by NAC.
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PMID:The organization of the human GSTP1-1 gene promoter and its response to retinoic acid and cellular redox status. 854 77

The Epstein-Barr virus-encoded protein BZLF1 is a member of the basic leucine zipper (bZip) family of transcription factors. Like several other members of the bZip family, transcriptional activity of BZLF1 is modulated by retinoic acid receptors (RARs). We present evidence that the RAR alpha and BZLF1 can reciprocally repress each other's transcriptional activation by a newly discovered mechanism. Analysis of RAR alpha mutants in transfection studies reveals that the DNA binding domain is sufficient for inhibition of BZLF1 activity. Analysis of BZLF1 mutants indicates that both the coiled-coil dimerization domain and a region containing the transcriptional activation domain of BZLF1 are required for transrepression. Coimmunoprecipitation experiments demonstrate physical interactions between RAR alpha and BZLF1 in vivo. Furthermore, glutathione S-transferase-pulldown assays reveal that these protein-protein interactions are mediated by the coiled-coil dimerization domain of BZLF1 and the DNA binding domain of RAR alpha. While RAR alpha is unable to recognize BZLF1 binding sites, the RAR alpha can be tethered to the DNA by forming a heteromeric complex with BZLF1 bound to DNA. Tethering RARs via protein-protein interactions onto promoter DNA suggest a mechanism through which RARs might gain additional levels of transcriptional regulation.
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PMID:Functional antagonism between the retinoic acid receptor and the viral transactivator BZLF1 is mediated by protein-protein interactions. 861 82

Unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of genes bearing thyroid hormone response elements in their promoters. Binding of hormonal ligand to the receptor releases the transcriptional silencing and leads to gene activation. Previous studies showed that the silencing activity of TR is located within the C-terminal ligand-binding domain (LBD) of the receptor. To dissect the role of the LBD in receptor-mediated silencing, we used a cell-free transcription system containing HeLa nuclear extracts in which exogenously added unliganded TRbeta repressed the basal level of RNA polymerase II-driven transcription from a thyroid hormone response element-linked template. We designed competition experiments with a peptide fragment containing the entire LBD (positions 145 to 456) of TRbeta. This peptide, which lacks the DNA-binding domain, did not affect basal RNA synthesis from the thyroid hormone response element-linked promoter when added to a cell-free transcription reaction mixture. However, the addition of the LBD peptide to a reaction mixture containing TRbeta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone. An LBD peptide harboring point mutations, which severely impair receptor dimerization, also inhibited efficiently the silencing activity of TR, indicating that the relief of repression by the LBD was not due to the sequestration of TR or its heterodimeric partner retinoid X receptor into inactive homo- or heterodimers. We postulate that the LBD peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extracts and is essential for efficient receptor-mediated gene repression. We have identified the region from positions 145 to 260 (the D domain) of the LBD as a potential binding site of the putative corepressor. We observed further that a peptide containing the LBD of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LBD may bind to the same corepressor activity as the TR LBD. Interestingly, the RAR LBD complexed with its cognate ligand, all-trans retinoic acid, failed to compete for transcriptional silencing by TRbeta, indicating that the association of the LBD with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supporting the existence of the corepressor activity in the HeLa nuclear extracts. Our studies demonstrated that the silencing activity of TR was greatly reduced in the nuclear extracts preincubated with immobilized, hormone-free glutathione S-transferase-LBD fusion proteins, indicating that the corepressor activity was depleted from these extracts through protein-protein interactions with the LBD. Similar treatment with immobilized, hormone-bound glutathione S-transferase-LBD, on the other hand, failed to deplete the corepressor activity from the nuclear extracts, indicating that ligand binding to the LBD disrupts its interaction with the corepressor. From these results, we propose that a corepressor binds to the LBD of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing. Ligand binding to TR results in the release of the corepressor from the LBD and triggers the reversal of silencing by allowing the events leading to gene activation to proceed.
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PMID:Transcriptional silencing by unliganded thyroid hormone receptor beta requires a soluble corepressor that interacts with the ligand-binding domain of the receptor. 862 57

Retinoic acid exerts many of its biological effects by interaction with heterocomplexes of nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). To further examine this interaction, a glutathione S-transferase (GST) fusion protein containing the ligand binding domain of human RXR alpha has been used to copurify the ligand binding domain of human RAR gamma by affinity chromatography over glutathione-agarose. Complexes of recombinant RAR-RXR ligand binding domains retaining full ligand binding capacity were purified, and their interactions with various retinoids were characterized by fluorometric titration and photoaffinity labeling. Analyses of the distribution of limiting amounts of [3H]-all-trans-retinoic acid between cytoplasmic retinoic acid binding proteins, CRABP-I and CRABP-II, and the purified heterocomplexes indicate that all-trans-retinoic acid binds with comparable affinity to CRABP-I and the heterocomplexes, but with approximately 10-fold less affinity to CRABP-II. The aromatic retinoid acitretin, which is used in the treatment of psoriasis, binds relatively poorly to the purified heterocomplexes, although it binds with high affinity to the CRABPs. Acitretin displaces [3H]-all-trans-retinoic acid from the CRABPs and increases retinoic acid occupancy of the heterocomplexes. These results suggest that certain retinoids could potentially perturb the distribution of endogenous retinoic acid between the CRABPs and the nuclear receptors and thus affect retinoid signaling. The purified recombinant complexes should provide a useful model system for further structural analysis of the dimerization interface between the RAR and RXR ligand binding domains.
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PMID:The isolation and characterization of purified heterocomplexes of recombinant retinoic acid receptor and retinoid X receptor ligand binding domains. 915 6

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors that belong to the steroid/thyroid hormone receptor (TR) superfamily and can repress the transcriptional activity of several target genes; however, the precise mechanism of this repression is unknown. Transfection of a Gal4 DNA-binding domain fused to the putative ligand-binding domain of COUP-TFI (Gal4-COUP-TFI) significantly represses the basal transcriptional activity of a reporter gene containing Gal4-binding sites. Cotransfection of COUP-TFI can relieve the Gal4-COUP-TFI repression in a dose-dependent manner. In contrast, COUP-TFI delta35, which lacks the repressor domain (the C-terminal 35 amino acids), fails to relieve this repression. This finding suggests that the repressor domain of COUP-TFI may squelch a limiting amount of corepressor in HeLa cells. In addition, increasing concentrations of TRbeta also can relieve the COUP-TFI repression in a hormone-sensitive manner. Similarly, overexpression of increasing concentration of COUP-TFI, but not COUP-TFI delta35, can squelch the silencing activity of the unliganded TRbeta. Collectively, these results indicate that COUP-TFI and TRbeta share a common corepressor(s) for their silencing activity. To determine which corepressor is involved in the COUP-TF-silencing activity, we used a yeast two-hybrid and in vitro GST pull-down assays to demonstrate that COUP-TFI can interact with the fragment of N-CoR (nuclear receptor-corepressor) encoding amino acids 921-2453 and the fragments of SMRT (silencing mediator for retinoic acid receptor and TR) encoding amino acids 29-564 and 565-1289, respectively. Interestingly, the fragment of SMRT encoding amino acids 1192-1495, which strongly interacts with TRbeta, interacts very weakly with COUP-TFI. Furthermore, overexpression of N-CoR or SMRT potentiates the silencing activity of COUP-TFI and can relieve the COUP-TFI-mediated squelching of Gal4-COUP-TFI activity. Therefore, our studies indicate that N-CoR and SMRT act as corepressors for the COUP-TFI silencing activity.
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PMID:Gene silencing by chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is mediated by transcriptional corepressors, nuclear receptor-corepressor (N-CoR) and silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT). 917 Dec 35

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was identified as a low abundance protein in bovine uterus that co-purified with estrogen receptor (ER) in a ligand-independent manner and was separated from the ER by its lower retention on estrogen response element (ERE)-Sepharose. In gel mobility shift assays, COUP-TF bound as an apparent dimer to ERE and ERE half-sites. COUP-TF bound to an ERE half-site with high affinity, Kd = 1.24 nM. In contrast, ER did not bind a single ERE half-site. None of the class II nuclear receptors analyzed, i.e. retinoic acid receptor, retinoid X receptor, thyroid receptor, peroxisome proliferator-activated receptor, or vitamin D receptor, were constituents of the COUP-TF.DNA binding complex detected in gel mobility shift assays. Direct interaction of COUP-TF with ER was indicated by GST "pull-down" and co-immunoprecipitation assays. The nature of the ER ligand influenced COUP-TF-ERE half-site binding. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), COUP-TF-half-site interaction decreased. Conversely, COUP-TF transcribed and translated in vitro enhanced the ERE binding of purified estradiol (E2)-liganded ER but not 4-OHT-liganded ER. Co-transfection of ER-expressing MCF-7 human breast cancer cells with an expression vector for COUP-TFI resulted in a dose-dependent inhibition of E2-induced expression of a luciferase reporter gene under the control of three tandem copies of EREc38. The ability of COUP-TF to bind specifically to EREs and half-sites, to interact with ER, and to inhibit E2-induced gene expression suggests COUP-TF regulates ER action by both direct DNA binding competition and through protein-protein interactions.
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PMID:Chicken ovalbumin upstream promoter-transcription factor interacts with estrogen receptor, binds to estrogen response elements and half-sites, and inhibits estrogen-induced gene expression. 939 81

BAG-1 (also known as RAP46) is an anti-apoptotic protein, which has been shown previously to interact with a number of nuclear hormone receptors, including receptors for glucocorticoid, estrogen, and thyroid hormone. We show here that BAG-1 also interacts with retinoic acid receptor (RAR). Gel retardation assays demonstrated that in vitro translated BAG-1 protein could effectively inhibit the binding of RAR but not retinoid X receptor (RXR) to a number of retinoic acid (RA) response elements (RAREs). A glutathione S-transferase-BAG-1 fusion protein also specifically bound RAR but not RXR. Interaction of BAG-1 and RAR could also be demonstrated by yeast two-hybrid assays. In transient transfection assays, co-transfection of BAG-1 expression plasmid inhibited the transactivation activity of RAR/RXR heterodimers but not RXR/RXR homodimers. When stably expressed in breast cancer cell lines, BAG-1 inhibited binding of RAR/RXR heterodimer to a number of RAREs and suppressed RA-induced growth inhibition and apoptosis. In addition, RA-induced suppression of Bcl-2 expression was abrogated by overexpression of BAG-1. These results demonstrate that BAG-1 can regulate retinoid activities through its interaction with RAR and suggest that elevated levels of BAG-1 protein could potentially contribute to retinoid resistance in cancer cells.
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PMID:Interaction of BAG-1 with retinoic acid receptor and its inhibition of retinoic acid-induced apoptosis in cancer cells. 964 62

Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor's silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.
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PMID:Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily. 1020 62

Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.
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PMID:Evidence for a novel cardiac-enriched retinoid X receptor partner. 1046 3

Ligand activation of retinoic acid receptors (RARs) involves coordinated changes in their interaction with coregulatory molecules. Binding of the agonist all-trans-retinoic acid to the RAR results in increased interaction with coactivator molecules as well as a decreased interaction with corepressor molecules. Thus, an all-trans-retinoic acid antagonist might function either by preventing agonist induction of such events or, additionally, by actively increasing repression via corepressor recruitment. We demonstrate that the repression of the transcriptional activity of a constitutively active RARgamma-VP-16 chimeric receptor by the inverse agonist AGN193109 requires a functional Co-R box and that binding of this ligand to RARgamma leads to an increased interaction with the corepressor N-CoR both in glutathione S-transferase pull-down and yeast two-hybrid analyses. Detection of nuclear receptor corepressor (N-CoR) association with RARgamma was greatly facilitated by inclusion of a RARE oligonucleotide in coimmunoprecipitation analyses, a result of an increase in association of the ternary complex consisting of RAR, RXR, and DNA. Similarly, this DNA-dependent increase in heterodimer formation likewise resulted in an increase in agonist-mediated recruitment efficiency of the coactivator SRC-1. Under conditions which favor ternary complex formation, a RAR neutral antagonist is distinguished from an inverse agonist with respect to corepressor recruitment as is a RAR partial agonist distinguished from an agonist with respect to coactivator recruitment. These results indicate that it is possible to design RAR ligands with distinct recruitment capabilities for coregulators, both coactivators as well as corepressors. In addition, using this recruitment assay, we show that SRC-1 and the related coactivator molecule ACTR associate with the ternary complex via utilization of different helical motifs within their conserved receptor interaction domains.
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PMID:Recruitment of nuclear receptor corepressor and coactivator to the retinoic acid receptor by retinoid ligands. Influence of DNA-heterodimer interactions. 1077 2


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