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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate beta-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.
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PMID:Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors. 910 40

Retinoid-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor superfamily. To study its physiological role we generated null-mutant mice by targeted insertion of a lacZ reporter gene encoding the enzyme beta-galactosidase. In heterozygous RORalpha+/- mice we found beta-galactosidase activity, indicative of RORalpha protein expression, confined to the central nervous system, skin and testis. In the central nervous system, the RORalpha gene is expressed in cerebellar Purkinje cells, the thalamus, the suprachiasmatic nuclei, and retinal ganglion cells. In skin, RORalpha is strongly expressed in the hair follicle, the epidermis, and the sebaceous gland. Finally, the peritubular cells of the testis and the epithelial cells of the epididymis also strongly express RORalpha. Recently, it was reported that the ataxic mouse mutant staggerer (sg/sg) is caused by a deletion in the RORalpha gene. The analysis of the cerebellar and the behavioral phenotype of homozygous RORalpha-/- mice proves identity to sg/sg mice. Although the absence of RORalpha causes dramatic developmental effects in the cerebellum, it has no apparent morphological effect on thalamus, hypothalamus, and retina. Similarly, testis and skin of RORalpha-/- mice display a normal phenotype. However, the pelage hair of both sg/sg and RORalpha-/- is significantly less dense and when shaved shows reluctance to regrow.
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PMID:staggerer phenotype in retinoid-related orphan receptor alpha-deficient mice. 952 Apr 75

Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid synthesized in the human adrenal cortex and serving as a biosynthetic precursor to testosterone and 17beta-estradiol. Despite the fact that it is one of the most abundant steroid hormones in circulation, the physiological role of DHEA in humans remains unclear. The action of DHEA itself, such as its interactions with receptors and nuclear transcription factors, is not well understood, and a specific DHEA receptor has yet to be identified. Although the activity of DHEA can be due to its metabolism into androgens and estrogens, DHEA has been shown to interact with the androgen receptor and the estrogen receptor (ER) in vitro. We demonstrate in this study that DHEA (3beta-Hydroxy-5alpha-androstan-17-one) inhibits 17beta-estradiol (E2) binding to its receptor in vivo in yeast. DHEA stimulates human ER dimerization in yeast, as determined by ER fusion protein interactions, GAL4 reconstitution and subsequent measurement of increased beta-galactosidase activity. DHEA causes an increase in estrogen response element-dependent beta-galactosidase activity, demonstrating that the ER dimer induced by DHEA is transcriptionally active, but at a concentration of DHEA about 1000 times greater than E2. Inclusion of the nuclear receptor co-activator RIP140 in the yeast enhances ER transactivation by DHEA or E2 in a ligand-dependent manner; moreover, only in the presence of RIP140 is DHEA able to stimulate beta-galactosidase activity to levels similar to those achieved by E2. Ligand-receptor interaction for other C19-steroids was also examined. While 5-androstene-3beta, 17beta-diol (ADIOL) displayed estrogenic activity in this system, 4-androstene-17-dione (androstenedione) and 4-androstene-17beta-ol,3-one (testosterone) did not. We have investigated whether DHEA can interact with the human ER in vivo. Our findings demonstrate a mechanism by which DHEA interacts directly with estrogen signaling systems; however, because DHEA is several orders of magnitude less potent than E2 in this system, we conclude that it essentially is not an estrogen agonist.
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PMID:Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in yeast. 980 58

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.
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PMID:Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis. 1062 44

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2(+/-) male mice we found that beta-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.
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PMID:Spermatogenesis and testis development are normal in mice lacking testicular orphan nuclear receptor 2. 1205 74

The hypothesis that cross-talk between membrane-active beta-adrenergic agonists and estrogens includes beta-adrenergic modulation of estrogen receptor (ER)-regulated gene expression was investigated. Vascular smooth muscle-derived A7r5 cells were transfected with an ERalpha expression plasmid (pCR3.1-hERalpha), the estrogen response element (ERE)-linked reporter pERE-E1b-luciferase (ERE-Luc), and pCMV-beta-galactosidase using a lysine-conjugated adenovirus transfection method. Hormone or agonist treatment and harvest followed 6 hours and 24 hours later, respectively. Treatment with 17beta-estradiol (E(2), 1 nmol/L) significantly stimulated ERE-Luc activity. Isoproterenol (10-9 to 10-6 mol/L) treatment alone did not stimulate ERE-Luc activity. Cotreatment with both E(2) and isoproterenol resulted in complete inhibition of E(2)-stimulated ERE-Luc activity. This isoproterenol effect was prevented by the beta-adrenergic antagonist propanolol (10-6 mol/L). Adrenomedullin treatment in these cells (1-50 nmol/L) did not inhibit ER/ERE-Luc activity, whether in the presence or absence of E(2). Moreover, isoproterenol did not affect vitamin D-stimulated VDRE-Luc expression, indicating that the inhibitory effect of isoproterenol on E(2)-directed ERE-Luc expression is specific among nuclear transcription factor receptors. Moreover, in MCF-7 breast cancer cells, there was no effect of isoproterenol on ER/ERE-directed transcription in the absence or presence of E(2), demonstrating tissue specificity of this isoproterenol effect. These studies demonstrate cross-talk between the beta-adrenergic agonist isoproterenol and ER-directed reporter gene expression in A7r5 cells. Furthermore, this cross-talk is specific with respect to agonist, nuclear receptor species, and cell type. These observations may have important implications both for the use of beta-adrenergic agents to treat hypertension and for possible gender-related differences in cardiovascular regulation.
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PMID:Cross-talk between beta-adrenergic stimulation and estrogen receptors: isoproterenol inhibits 17beta-estradiol-induced gene transcription in A7r5 cells. 1288 32

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.
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PMID:A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS. 1869 91

Steroid hormone receptor-mediated reporter assays in the budding yeast Saccharomyces cerevisiae have been an invaluable tool for the identification and functional characterization of steroid hormone receptor-associated chaperones and cochaperones. This chapter describes a hormone-inducible androgen receptor-mediated beta-galactosidase reporter assay in yeast. In addition, the immunophilin FKBP52 is used as a specific example of a receptor-associated cochaperone that acts as a positive regulator of receptor function. With the right combination of receptor and cochaperone expression plasmids, reporter plasmid, and ligand, the assay protocol described here could be used to functionally characterize a wide variety of nuclear receptor-cochaperone interactions. In addition to the functional characterization of receptor regulatory proteins, a modified version of this assay is currently being used to screen compound libraries for selective FKBP52 inhibitors that represent attractive therapeutic candidates for the treatment of steroid hormone receptor-associated diseases.
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PMID:Yeast-based reporter assays for the functional characterization of cochaperone interactions with steroid hormone receptors. 1911 43

Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is an orphan member of the nuclear receptor superfamily and is required for normal mouse embryonic development. In adult mice, NR6A1 is predominantly expressed in spermatogenic cells and growing oocytes of the gonads and has a role in female reproduction by modulating the transcription of the oocyte-specific genes bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9). In our goal to further understand the functional role of NR6A1 during postnatal development, we generated a Nr6a1:beta-galactosidase (LacZ) knockin reporter (Nr6a1(LacZ/+)) mouse line in which the Nr6a1:LacZ fusion gene was expressed and then characterized Nr6a1 expression in these reporter mice by performing LacZ staining. Our RT-PCR analyses showed that Nr6a1 was expressed in a variety of somatic tissues (e.g., oviduct and lung) other than gonads of normal adult mice. In adult Nr6a1(LacZ/+) mice, robust LacZ staining was observed in the gametes of gonads. Strong positive LacZ staining was also observed in the sperm of the epididymis, epithelial cells of the oviduct, and bronchioles within the lung. In adult Nr6a1(LacZ/+) mice, positive LacZ staining was observed in other somatic tissues, including hippocampus, cerebral cortex, cerebellum, and thalamus of brain; pars intermedia and pars anterior of pituitary; parathyroid; and islet of pancreas. NR6A1 expression in sperm within the epididymis, epithelial cells in the oviduct, and bronchioles of the lung was further confirmed by immunohistochemical studies. Nr6a1 is expressed not only in the germ cells of mouse gonads but also in a variety of somatic tissues, including epididymis, oviduct, brain, and pituitary. The extra-germ cell expression of NR6A1 makes it a less attractive contraceptive.
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PMID:Extra-germ cell expression of mouse nuclear receptor subfamily 6, group A, member 1 (NR6A1). 1916 81

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