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)

The tumor suppressor protein p53 exerts its cell cycle-regulatory effects through its ability to function as a sequence-specific DNA-binding transcription factor. Herein, we show that p53 physically interacts with specific subregions of steroid receptor coactivator-1 (SRC-1) and its family members, p/CIP (p300/CBP interacting protein), xSRC-3, and AIB1 (amplified in breast cancer), originally isolated as transcription coactivators of nuclear receptors, as demonstrated by the yeast and mammalian two-hybrid tests as well as glutathione S-transferase pull-down assays. Interestingly, cotransfection of HeLa cells with SRC-1- or p/CIP expression vector potentiated the p53-mediated transactivation, whereas AIB1 and xSRC-3 were repressive. All of these SRC-1 members, however, similarly stimulated transactivation mediated by nuclear receptors and AP-1, as previously described. These results suggest that SRC-1 and its family members may differentially modulate the p53 transactivation in vivo.
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PMID:Steroid receptor coactivator-1 and its family members differentially regulate transactivation by the tumor suppressor protein p53. 1055 85

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

The nuclear peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily and acts as a ligand-dependent transcription factor mediating adipocyte differentiation, cell proliferation and inflammatory processes, and modulation of insulin sensitivity. Members of the 160-kDa protein (SRC-1/TIF2/AIB-1) family of coactivators, CBP/p300 and TRAP220/DRIP205, are shown to interact directly with PPARgamma and potentiate nuclear receptor transactivation function in a ligand-dependent fashion. Because PPARgamma ligands exert partially overlapping but distinct subsets of biological action through PPARgamma binding, we wished to examine whether interactions between PPARgamma and known coactivators were induced to the same extent by different classes of PPARgamma ligand. The natural ligand 15-deoxy-Delta12,14-prostaglandin J(2) induced PPARgamma interactions with all coactivators tested (SRC-1, TIF2, AIB-1, p300, TRAP220/DRIP205) in yeast and mammalian two-hybrid assays, as well as in a glutathione S-transferase pull-down assay. However, under the same conditions troglitazone, a synthetic PPARgamma ligand that acts as an antidiabetic agent, did not induce PPARgamma interactions with any of the coactivators. Our findings suggest that ligand binding may alter PPARgamma structure in a ligand type-specific way, resulting in distinct PPARgamma-coactivator interactions.
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PMID:Ligand type-specific interactions of peroxisome proliferator-activated receptor gamma with transcriptional coactivators. 2414 11

An androgen receptor (AR) interacting protein was isolated from a HeLa cell complementary DNA library by two-hybrid screening in yeast using the AR DNA and ligand binding domains [amino acids (aa) 481-919] as bait. AR binding of the protein in yeast was dependent on the presence of testosterone or dihydrotestosterone (DHT). The isolated protein is identical to thyroid receptor activator molecule TRAM-1 but lacking aa 1-458. TRAM-1 is a steroid receptor coactivator-3 (SRC-3) subtype. In affinity matrix assays, 35S-labeled TRAM-1 bound the GST-AR ligand binding domain (aa 624-919) and GST-AR N-terminal and DNA binding domains (aa 1-660), but not the GST-AR DNA binding domain (aa 544-634) alone. Coexpression of TRAM-1 increased DHT-dependent AR transactivation 5-fold and constitutive activity of AR (aa 1-660) N-terminal and DNA-binding domains increased 9-fold. Full-length TRAM-1 (aa 1-1424) and the partial (aa 459-1424) were AR and GR coactivators as was SRC-1. In human testis, immunostaining of SRC-3 colocalized with AR in nuclei of Sertoli cells and peritubular myoid cells, indicating it could function as an AR coactivator in these cells. SRC-3 was also present in nuclei of spermatogenic cells where AR was not expressed, suggesting it might also be a coactivator with other nuclear receptors that regulate spermatogenesis.
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PMID:Thyroid receptor activator molecule, TRAM-1, is an androgen receptor coactivator. 1096 17

Phytoestrogens are assumed to affect the endocrine system of animal species similarly to other man-made endocrine disrupters and to exert their effects through estrogen receptors, specifically ER(alpha) and ERbeta. However, these molecular mechanisms are not fully understood. In this study, 19 phytochemicals were surveyed for agonist and antagonist activities of ER(alpha) and ERbeta using an ERE-luciferase reporter assay. The results showed that ferutinine is an agonist for ER(alpha) and an agonist/antagonist for ERbeta, tschimgine is an agonist for both ER(alpha) and ERbeta, and tschimganidine is an agonist for only ER(alpha). Ferutinine and tschimganidine are sesquiterpenoids, and tschimgine is a monoterpenoid derived from the Umbelliferae family. A competitive binding assay showed that ferutinine has higher binding affinities than tamoxifen for both ERs. Co-transfections of coactivators such as SRC-1, TIF2, AIB1, and TRAP220 in 293T cells and use of the luciferase assay revealed that TRAP220 failed to enhance the transcription mediated by ERbeta in the presence of ferutinine. Moreover, a GST pull-down assay showed that TRAP220 marginally bound to ERbeta ligand binding domain in the presence of ferutinine. These results suggest that the conformation of ferutinine-liganded ERbeta is difficult for TRAP220 to recognize. Taken together, this suggests that some terpenoids can modulate estrogen signaling as ER subtype-selective phytoestrogens similar to SERMs (selective estrogen receptor modulators).
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PMID:Terpenoids found in the umbelliferae family act as agonists/antagonists for ER(alpha) and ERbeta: differential transcription activity between ferutinine-liganded ER(alpha) and ERbeta. 1184 12

The aryl hydrocarbon receptor complex heterodimeric transcription factor, comprising the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) domain aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, mediates the toxic effects of TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin). The molecular events underlying TCDD-inducible gene activation, beyond the activation of the AHRC, are poorly understood. The SRC-1/NCoA-1, NCoA-2/GRIP-1/TIF-2, and p/CIP/AIB/ACTR proteins have been shown to act as mediators of transcriptional activation. In this report, we demonstrate that SRC-1, NCoA-2, and p/CIP are capable of independently enhancing TCDD-dependent induction of a luciferase reporter gene by the AHR/ARNT dimer. Furthermore, injection of anti-SRC-1 or anti-p/CIP immunoglobulin G into mammalian cells abolishes the transcriptional activity of a TCDD-dependent reporter gene. We demonstrate by coimmunoprecipitation and by a reporter gene assay that SRC-1 and NCoA-2 but not p/CIP are capable of interacting with ARNT in vivo after transient transfection into mammalian cells, while AHR is capable of interacting with all three coactivators. We confirm the interactions of ARNT and AHR with SRC-1 with immunocytochemical techniques. Furthermore, SRC-1, NCoA-2, and p/CIP all associate with the CYP1A1 enhancer region in a TCDD-dependent fashion, as demonstrated by chromatin immunoprecipitation assays. We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1. This indicates a novel mechanism of action for SRC-1. SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain. Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation.
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PMID:Recruitment of the NCoA/SRC-1/p160 family of transcriptional coactivators by the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator complex. 1202 42

Nuclear mitotic apparatus protein-retinoic acid receptor alpha (NuMA-RARalpha) is the fourth of five fusion proteins identified in acute promyelocytic leukemia (APL) patients. The molecular basis for its oncogenic activity has not been delineated. In gel-shift assays, NuMA-RARalpha bound to retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXRalpha. The binding profile of NuMA-RARalpha to a panel of RAREs was very similar to PML-RARalpha and PLZF-RARalpha. In transient transfection assays using HepG2 cells, NuMA-RARalpha inhibited wild-type RARalpha transcriptional activity, while it augmented STAT3 transcriptional activity. In GST-pull down experiments, NuMA-RARalpha formed a complex with the corepressor SMRT, was released from the NuMA-RARalpha/SMRT complexes by all-trans retinoic acid (ATRA) at 10(-7)-10(-6) M and became associated with the coactivator TRAM-1 at 10(-8) M ATRA. Studies comparing NuMA-RARalpha with NuMA-RARalpha(deltaCC) demonstrated that the dimerization or alpha-helical coiled-coil domain of NuMA was required for homodimer formation, transcriptional repression of wild-type RARalpha, transcriptional activation of STAT3, and stability of the NuMA-RARalpha/SMRT complex. Confocal fluorescent microscopy of HeLa cells was performed following transient expression of cyan fluorescent protein (CFP)-tagged proteins and incubation of cells with or without ATRA. Within the nucleus, CFP-NuMA-RARalpha exhibited a speckled pattern identical to that observed in cells transfected with CFP-NuMA. Furthermore, CFP-NuMA-RARalpha colocalized with yellow fluorescent protein-tagged (YFP)-NuMA. In contrast, CFP-NuMA-RARalpha(deltaCC) exhibited a diffuse granular pattern within the nucleus, similar to RARalpha. These results indicate that the dimerization domain of NuMA-RARalpha is critical for each of the known oncogenic activities of NuMA fusion proteins as well as its sequestration to nuclear sites normally occupied by NuMA and is distinct from RARalpha.
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PMID:Essential role for the dimerization domain of NuMA-RARalpha in its oncogenic activities and localization to NuMA sites within the nucleus. 1258 66

Assessment of the risk of human exposure to man-made chemicals that bind to hormone receptors has emerged as a major public health issue. Among hormone receptors, nuclear receptors tend to be targets of xenobiotics because their endogenous ligands are small, fat-soluble molecules. Nuclear receptors are ligand-inducible transcriptional factors and regulate the transcriptional activity of various target genes. At the start of the initiation step of transcription, nuclear receptors interact with coactivators (TIF2, SRC1, ACTR, CBP/p300, etc.) in an agonist-dependent manner. Using the interaction of the nuclear receptor with a coactivator, we have developed a novel rapid ligand in vitro screening method that is easy to use and has high sensitivity. This method, called by us the CoA-BAP system, is applicable to most nuclear receptors and is suitable for high-throughput screening because the entire experimental operation can be carried out on a microplate. We used human TIF2 as a coactivator including LXXLL motifs expressed in Escherichia coli as a fusion protein with BAP and nuclear receptor LBD expressed in E. coli as a fusion protein with GST. On a GSH-coupled microplate these proteins were incubated with chemicals and the protein-protein interactions were detected as alkaline phosphatase activity. To date we have examined seven nuclear receptors (ERalpha/beta, TRalpha, RARalpha/gamma, RXRalpha,and VDR) and confirmed that the method works well.
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PMID:Basis of a high-throughput method for nuclear receptor ligands. 1286 36

Resistance to the action of vitamin D (D) occurs in response to tumor necrosis factor-alpha (TNF-alpha), an effect mediated by nuclear factor kappa B (NfkappaB). To determine the mechanism of NfkappaB inhibition of D-stimulated transcription, chromatin immunoprecipitation assays (CHIP) were done in osteoblastic ROS 17/2.8 cells that had been treated with TNF-alpha or transfected with the p65 subunit of NfkappaB. These treatments caused stable incorporation of p65 into the transcription complex bound to the vitamin D response element (VDRE) of the osteocalcin promoter. Deletion analysis of p65 functional domains revealed that the p65 N-terminus and a midmolecular region were both required for the inhibitory action of p65. Pull-down assays were done using an immobilized glutathione S-transferase (GST)-VDR fusion protein to study the effect of p65 on VDR binding to steroid coactivator-1 (SRC-1), a major D-dependent coactivator. p65 inhibited VDR-SRC-1 binding in a dose-dependent manner. Mutations of p65 that abrogated the inhibitory effect on D-stimulated transcription also failed to inhibit VDR-SRC-1 binding. The inhibitory effect of p65 on VDR transactivation was not due to recruitment of a histone deacetylase (HDAC), since inhibition was not relieved by the HDAC inhibitors sodium butyrate or trichostatin A. Overexpression of SRC-1 or the general coactivators, Creb binding protein or SRC-3, also failed to relieve p65 inhibition of transcription. In addition, Chip assays revealed that TNF-alpha treatment prevented D recruitment of SRC-1 to the transcription complex. These results show that TNF-alpha inhibition of vitamin D-action includes stable integration of p65 in the VDR transcription complex. Once anchored to proteins within the complex, p65 disrupts VDR binding to SRC-1, thus decreasing the efficiency of D-stimulated gene transcription.
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PMID:Integration of the NfkappaB p65 subunit into the vitamin D receptor transcriptional complex: identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription. 1521 79

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.
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PMID:Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in VDR transactivation during keratinocyte differentiation. 1522 84


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