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)

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

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

Angiotensin (A) II plays a critical role in vascular remodeling, and its action is mediated by type 1 AII receptor (AT1R). Recently, 15-deoxy-(Delta)(12,14)-prostaglandin J(2) and thiazolidinediones have been shown to be ligands for peroxisome proliferator-activated receptor (PPAR)-gamma and activate PPAR-gamma. In the present work, we have studied the effect of PPAR-gamma on AT1R expression in rat vascular smooth muscle cells (VSMCs). We observed that: 1) endogenous AT1R expression was significantly decreased by PPAR-gamma ligands both at messenger RNA and protein levels, whereas AT1R messenger RNA stability was not affected; 2) AII-induced increase of (3)H-thymidine incorporation into VSMCs was inhibited by PPAR-gamma ligands; 3) rat AT1R gene promoter activity was significantly suppressed by PPAR-gamma ligands, and PPAR-gamma overexpression further suppressed the promoter activity; 4) transcriptional analyses using AT1R gene promoter mutants revealed that a GC-box-related sequence within the -58/-34 region of the AT1R gene promoter was responsible for the suppression; 5) Sp1 overexpression stimulated AT1R gene transcription via the GC-box-related sequence, which was inhibited by additional PPAR-gamma overexpression; 6) electrophoretic mobility shift assay suggested that Sp1 could bind to the GC-box-related sequence whereas PPAR-gamma could not; 7) antibody supershift experiments using VSMC nuclear extracts revealed that protein-DNA complexes formed on the GC-box-related sequence, which were decreased by PPAR-gamma coincubation, were mostly composed of Sp1; and 8) glutathione S-transferase pull-down assay revealed a direct interaction between PPAR-gamma and Sp1. Taken together, it is suggested that activated PPAR-gamma suppresses AT1R gene at a transcriptional level by inhibiting Sp1 via a protein-protein interaction. PPAR-gamma ligands, thus, may inhibit AII-induced cell growth and hypertrophy in VSMCs by AT1R expression suppression and possibly be beneficial for treatment of diabetic patients with hypertension and atherosclerosis.
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PMID:Transcriptional suppression of type 1 angiotensin II receptor gene expression by peroxisome proliferator-activated receptor-gamma in vascular smooth muscle cells. 1141 35

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.
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PMID:Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate. 1159 Jun 8

Small heterodimer partner (SHP, NR0B2) is an atypical orphan nuclear receptor that inhibits transcriptional activation by several other nuclear receptors. We recently reported that mutations in the SHP gene are associated with insulin resistance. In the present study, we demonstrated that the SHP gene is expressed in adipose tissues. A reporter gene assay showed that a gene product of SHP increased the transcriptional activation of peroxisome proliferator-activated receptor (PPAR) gamma. SHP-mediated activation of PPARgamma was observed both in the presence and absence of the ligand of PPARgamma. Immunoprecipitation and glutathione S-transferase pull-down assay showed that SHP directly bound to PPARgamma and competed with nuclear receptor corepressor for binding to PPARgamma. Serial deletion studies indicated that the C terminus of SHP is important for PPARgamma activation. Mutant SHP proteins, which are found in naturally occurring mutation, showed less enhancing activity for PPARgamma than wild-type SHP. Our results suggest that SHP may act as an endogenous enhancer of PPARgamma.
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PMID:Small heterodimer partner, an orphan nuclear receptor, augments peroxisome proliferator-activated receptor gamma transactivation. 1169 34

Although PGC-1 (peroxisome proliferator-activated receptor-gamma coactivator-1) has been previously shown to enhance thyroid hormone receptor (TR)/retinoid X receptor-mediated ucp-1 gene expression in a ligand-induced manner in rat fibroblast cells, the precise mechanism of PGC-1 modulation of TR function has yet to be determined. In this study, we show that PGC-1 can potentiate TR-mediated transactivation of reporter genes driven by natural thyroid hormone response elements both in a ligand-dependent and ligand-independent manner and that the extent of coactivation is a function of the thyroid hormone response element examined. Our data also show that PGC-1 stimulation of TR activity in terms of Gal4 DNA-binding domain fusion is strictly ligand-dependent. In addition, an E457A AF-2 mutation had no effect on the ligand-induced PGC-1 enhancement of TR activity, indicating that the conserved charged residue in AF-2 is not essential for this PGC-1 function. Furthermore, GST pull-down and mammalian two-hybrid assays demonstrated that the PGC-1 LXXLL motif is required for ligand-induced PGC-1/TR interaction. This agonist-dependent PGC-1/TR interaction also requires both helix 1 and the AF-2 region of the TR ligand-binding domain. Taken together, these results support the notion that PGC-1 is a bona fide TR coactivator and that PGC-1 modulates TR activity via a mechanism different from that utilized with peroxisome proliferator activator receptor-gamma.
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PMID:Requirement of helix 1 and the AF-2 domain of the thyroid hormone receptor for coactivation by PGC-1. 1175 19

Thromboxane (TX) A(2) exerts contraction and proliferation of vascular smooth muscle cells (VSMCs) via its specific membrane TX receptor (TXR), possibly leading to the progression of atherosclerosis. A nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-gamma, has recently been reported to be expressed in VSMCs. Here we examined a role of PPAR-gamma in TXR gene expression in VSMCs. PPAR-gamma ligands 15-deoxy-Delta(12,14)-prostaglandin J(2) and troglitazone reduced TXR mRNA expression levels as well as cell growth as assessed by [(3)H]thymidine incorporation. Transcriptional activity of the TXR gene promoter was suppressed with PPAR-gamma ligands, and the suppression was augmented further by PPAR-gamma overexpression. By deletion and mutation analyses, the transcription suppression was shown to be the result of a -22/-7 GC box-related sequence (upstream of transcription start site). Electrophoretic mobility shift assays also showed that the sequence was bound by Sp1 but not by PPAR-gamma, and the formation of a Sp1 small middle dotDNA complex was inhibited either by coincubation with PPAR-gamma or PPAR-gamma ligand treatment of VSMCs. Moreover, glutathione S-transferase pull-down assays demonstrated a direct interaction between PPAR-gamma and Sp1. In conclusion, PPAR-gamma suppresses TXR gene transcription via an interaction with Sp1. PPAR-gamma may possibly have an antiatherosclerotic action by inhibiting TXR gene expression in VSMCs.
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PMID:Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-gamma via an interaction with Sp1 in vascular smooth muscle cells. 1177 1

The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPARalpha-specific activator. Phytol induced the increase in PPARalpha-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPARalpha. Moreover, the addition of phytol upregulated the expression of PPARalpha-target genes at both mRNA and protein levels in PPARalpha-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPARalpha ligand and that it stimulates the expression of PPARalpha-target genes in intact cells. Because PPARalpha activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism.
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PMID:Phytol directly activates peroxisome proliferator-activated receptor alpha (PPARalpha) and regulates gene expression involved in lipid metabolism in PPARalpha-expressing HepG2 hepatocytes. 1620 84

The peptide hormone glucagon stimulates hepatic glucose output, and its levels in the blood are elevated in type 2 diabetes mellitus. The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has essential roles in glucose homeostasis, and thiazolidinedione PPARgamma agonists are clinically important antidiabetic drugs. As part of their antidiabetic effect, thiazolidinediones such as rosiglitazone have been shown to inhibit glucagon gene transcription through binding to PPARgamma and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene. However, how thiazolidinediones and PPARgamma inhibit PAX6 activity at the glucagon promoter remained unknown. After transient transfection of a glucagon promoter-reporter fusion gene into a glucagon-producing pancreatic islet alpha-cell line, ligand-bound PPARgamma was found in the present study to inhibit glucagon gene transcription also after deletion of its DNA-binding domain. Like PPARgamma ligands, also retinoid X receptor (RXR) agonists inhibited glucagon gene transcription in a PPARgamma-dependent manner. In glutathione transferase pull-down assays, the ligand-bound PPARgamma-RXR heterodimer bound to the transactivation domain of PAX6. This interaction depended on the presence of the ligand and RXR, but it was independent of the PPARgamma DNA-binding domain. Chromatin immunoprecipitation experiments showed that PPARgamma is recruited to the PAX6-binding proximal glucagon promoter. Taken together, the results of the present study support a model in which a ligand-bound PPARgamma-RXR heterodimer physically interacts with promoter-bound PAX6 to inhibit glucagon gene transcription. These data define PAX6 as a novel physical target of PPARgamma-RXR.
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PMID:A peroxisome proliferator-activated receptor gamma-retinoid X receptor heterodimer physically interacts with the transcriptional activator PAX6 to inhibit glucagon gene transcription. 1796 86

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is a model of hyperphagic obesity in which the animals retain the desire to run voluntarily. Running wheels were provided for 4-wk-old OLETF rats for 16 wk before they were killed 5 h (WL5), 53 h (WL53), or 173 h (WL173) after the wheels were locked. Sedentary (SED) OLETF rats that were not given access to running wheels served as age-matched cohorts. Epididymal fat pad mass, adipocyte volume, and adipocyte number were 58%, 39%, and 47% less, respectively, in WL5 than SED rats. Contrary to cessation of daily running in Fischer 344 x Brown Norway rats, epididymal fat did not increase during the first 173 h of running cessation in the OLETF runners. Serum insulin and glucose levels were 77% and 29% less, respectively, in WL5 than SED rats. Oil red O staining for intramyocellular lipid accumulation was not statistically different among groups. However, lipid peroxidation levels, as determined by total trans-4-hydroxy-2-nonenal (4-HNE) and 4-HNE normalized to oil red O, was higher in epitrochlearis muscles of SED than WL5, WL53, and WL173 rats. mRNA levels of glutathione S-transferase-alpha type 4, an enzyme involved in cellular defense against electrophilic compounds such as 4-HNE, were higher in epitrochlearis muscle of WL53 than WL173 and SED rats. In contrast, 4-HNE levels in omental fat were unaltered. Epitrochlearis muscle palmitate oxidation and relative transcript levels for peroxisome proliferator-activated receptor-delta and peroxisome proliferator-activated receptor-gamma coactivator type 1 were surprisingly not different between runners and SED rats. In summary, voluntary running was associated with lower levels of lipid peroxidation in skeletal muscle without significant changes in intramyocellular lipids or mitochondrial markers in OLETF rats at 20 wk of age. Therefore, even in a genetic animal model of extreme overeating, daily physical activity promotes improved health of skeletal muscle.
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PMID:Exercise-induced attenuation of obesity, hyperinsulinemia, and skeletal muscle lipid peroxidation in the OLETF rat. 1807 66


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