Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.
Mol Endocrinol 2002 Jun
PMID:Activity of the GR in G2 and mitosis. 1204 20

Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100-270-fold increases in k(cat)/K(m)(Phe) and had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001<or=p<or=0.008) concentration of mutations occurs in a subset of positions (set AAT-TAT) that is conserved (>or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection TAT) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT-TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15 A of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.
J Mol Biol 2003 Mar 28
PMID:How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase. 1263 55

The homodimeric, pyridoxal 5'-phosphate (PLP)-dependent enzyme glutamine transaminase K/cysteine conjugate beta-lyase (GTK/beta-lyase) has been implicated in the bioactivation of chemopreventive compounds. This paper describes the first homology model of rat renal GTK/beta-lyase and its active site residues, deduced from molecular dynamics (MD) simulations of the binding mode of 13 structurally diverse cysteine S-conjugates and amino acids after Amber-parametrization of PLP. Comparison with Thermus thermophilus aspartate aminotransferase (tAAT) and Trypanosoma cruzi tyrosine aminotransferase (tTAT), used as templates for modeling GTK/beta-lyase, showed that the PLP-binding site of GTK/beta-lyase is highly conserved. Binding of the ligand alpha-carboxylate-group occurred via the conserved residues Arg(432) and Asn(219), and Asn(50) and Gly(70). Two pockets accommodated the various ligand side chains. A small pocket, located directly above PLP, was of a highly hydrophobic and aromatic character. A larger pocket, formed partly by the substrate access channel, was more hydrophilic and notably involved the salt bridge partners Glu(54) and Arg(99*) (* denotes the other subunit). Ligand-binding residues included Leu(51), Phe(71), Tyr(135), Phe(373) and Phe(312*), and pi-stacking interactions were often observed. Tyr(135) and Asn(50) were prominent in hydrogen bonding with the sulfur-atom of cysteine S-conjugates. The observed binding mode of the ligands corresponded well with their experimentally determined inhibitory potency toward GTK/beta-lyase. The current homology model thus provides a starting point for further validation of the role of active site residues in ligand-binding by means of mutagenesis studies. Ultimately, insight in the binding of ligands to GTK/beta-lyase may result in the rational design of new ligands and selective inhibitors.
J Mol Graph Model 2003 Sep
PMID:Modeling and molecular dynamics of glutamine transaminase K/cysteine conjugate beta-lyase. 1279 91

The xenobiotic-mediated induction of three major human liver cytochrome P450 genes, CYP2B6, CYP2C9, and CYP3A4, is known to be regulated by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). CAR and PXR are regulated, at least in part, by the glucocorticoid receptor (GR) and the hypothesis of a signal transduction cascade GR-[CAR/PXR]-P450 has been proposed. This study was aimed at testing this hypothesis in primary human hepatocytes by using the tubulin network disrupting agent colchicine. Colchicine (COL) decreased both basal and rifampicin- and phenobarbital-inducible expression of CYP2B6, CYP2C8/9, and CYP3A4. A parallel down-regulation of mRNA expression of CAR, PXR, and tyrosine aminotransferase, a prototypic gene directly regulated by GR, was observed. COL affected neither the level of GR mRNA nor ligand binding to GR. To evaluate the effect of colchicine on GR-mediated gene transactivation, HeLa cells stably or transiently transfected with a GR-responsive element-dependent luciferase reporter gene were used. COL decreased the dexamethasone-induced luciferase expression in stably transfected cell line by 50%, whereas GR transactivation in transiently transfected cells was not affected by COL. In contrast, ligand-dependent GR translocation in the human embryonic kidney 293 cell line transiently transfected with GFP-GR was inhibited by COL. We conclude that alteration of the signal transduction mediated through the GR-[CAR/PXR]-P450 cascade by colchicine is responsible for the down-regulation of CYP2C9 and CYP3A4, implicating cytoskeleton as necessary for correct functioning of this cascade under physiological conditions.
Mol Pharmacol 2003 Jul
PMID:Colchicine down-regulates cytochrome P450 2B6, 2C8, 2C9, and 3A4 in human hepatocytes by affecting their glucocorticoid receptor-mediated regulation. 1281 72

Microarray technology was evaluated for usefulness in assessing relationships between serum corticosterone and hepatic gene expression. Nine pairs of female Swiss mice were chosen to provide a wide range of serum corticosterone ratios; cDNA microarray analysis (approximately 8000 genes) was performed on their livers. A statistical method based on calculation of 99% confidence intervals discovered 32 genes which varied significantly among the livers. Five of these ratios correlated significantly with serum corticosterone ratio, including tyrosine aminotransferase, stress-induced protein, pleiotropic regulator 1 and insulin-like growth factor-binding protein-1; the latter has a potential role in cancer development. Secondly, linear regression of gene expression vs corticosterone ratios was screened for those with r> or =0.8 (P<0.01), yielding 141 genes, including some known to be corticosterone regulated and others of interest as possible glucocorticoid targets. Half of these significant correlations involved data sets where no microarray ratio exceeded +/- 1.5. These results showed that microarray may be used to survey tissues for changes in gene expression related to serum hormones, and that even small changes in expression can be of statistical significance in a study with adequate numbers of replicate samples.
J Mol Endocrinol 2004 Feb
PMID:Expressions of hepatic genes, especially IGF-binding protein-1, correlating with serum corticosterone in microarray analysis. 1476 7

To gain a better understanding of the nature of active chromatin in mammals, we have characterized in living cells the various chromatin modification events triggered by the glucocorticoid receptor (GR) at the rat tyrosine aminotransferase gene. GR promotes a local remodeling at a glucocorticoid-responsive unit (GRU) located 2.5 kb upstream of the transcription start site, creating nuclease hypersensitivity that encompasses 450 bp of DNA. Nucleosomes at the GRU occupy multiple frames that are remodeled without nucleosome repositioning, showing that nucleosome positioning is not the key determinant of chromatin accessibility at this locus. Remodeling affects nucleosomes and adjacent linker sequences, enhancing accessibility at both regions. This is associated with decreased interaction of both the linker histone H1 and the core histone H3 with DNA. Thus, our results indicate that nucleosome and linker histone removal rather than nucleosome repositioning is associated with GR-triggered accessibility. Interestingly, GR induces hyperacetylation of histones H3 and H4, but this is not sufficient either for remodeling or for transcriptional activation. Finally, our data favor the coexistence of several chromatin states within the population, which may account for the previously encountered difficulties in characterizing unambiguously the active chromatin structure in living cells.
Mol Cell Biol 2004 Sep
PMID:Nature of the accessible chromatin at a glucocorticoid-responsive enhancer. 1534 52

The human glucocorticoid receptor (hGR) beta, a splicing variant of the classic receptor hGRalpha, functions as a dominant-negative inhibitor of hGRalpha. We explored the mechanism(s) underlying this effect of hGRbeta by evaluating the interactions of this isoform with known steroid receptor coactivators. We found that hGRbeta suppressed the transcriptional activity of both activation function (AF)-1 and AF-2 of hGRalpha, indicating that hGRbeta may exert its dominant-negative effect by affecting the function of coactivators that are attracted to these transactivation domains. hGRbeta bound to one of the p160 coactivators, the glucocorticoid receptor-interacting protein 1 (GRIP1) via its preserved AF-1 but not via its defective AF-2 in vitro. In a chromatin immunoprecipitation assay, hGRbeta prevented coprecipitation of GRIP1 with hGRalpha tethered to glucocorticoid response elements of the endogenous tyrosine aminotransferase promoter, whereas deletion of the AF-1 of hGRbeta abolished this effect. In further experiments, overexpression of GRIP1 attenuated the suppressive effect of hGRbeta on hGRalpha-mediated transactivation of the mouse mammary tumor virus promoter. Competition for binding to glucocorticoid response elements or heterodimerization with hGRalpha via the D loop dimerization interface occurred, but they were not necessary for the suppressive effect of hGRbeta on the transcriptional activity of hGRalpha. Our findings suggest that hGRbeta suppresses the transcriptional activity of hGRalpha by competing with hGRalpha for binding to GRIP1, and possibly other p160 coactivators, through its preserved AF-1. These findings suggest that participation of hGRbeta in the formation of a coactivator complex renders this complex ineffective.
Mol Endocrinol 2005 Jan
PMID:The human glucocorticoid receptor (hGR) beta isoform suppresses the transcriptional activity of hGRalpha by interfering with formation of active coactivator complexes. 1545 52

The effects of rat-specific hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), mouse-specific hepatocarcinogen ortho-aminoazotoluene (OAT), non-species-specific hepatocarcinogen diethylnitrosamine (DENA), and non-carcinogenic 4'-methyl-4-dimethylaminoazobenzene (4'-MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA-binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen-susceptible and -resistant animals. Species-specific hepatocarcinogens 3'-MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non-carcinogenic 4'-MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA-binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species-specific effects of OAT and of 3'-MeDAB on HNF3 DNA-binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32-0.47 g/mL interval of ammonium sulfate concentration. In contrast, non-specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.
Mol Carcinog 2005 Dec
PMID:Species-specific effects of the hepatocarcinogens 3'-methyl-4-dimethyl-aminoazobenzene and ortho-aminoazotoluene in mouse and rat liver. 1626 30

It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded.
J Steroid Biochem Mol Biol 2006 Feb
PMID:Transfection with steroid-responsive reporter constructs shows glucocorticoid rather than androgen responsiveness in cultured Sertoli cells. 1638 47

Tyrosinemia type II or Richner-Hanhart Syndrome (RHS) is an autosomal recessive disorder characterized by keratitis, palmoplantar keratosis, mental retardation, and elevated blood tyrosine levels. The disease is due to a deficiency of hepatic cytosolic tyrosine aminotransferase (TATc), an enzyme involved in the tyrosine catabolic pathway. Because of the high rate of consanguinity this disorder seems to be relatively common among the Arab and Mediterranean populations. RHS is characterized by inter and intrafamilial phenotypic variability. A large spectrum of mutations within TATc gene has been shown to be responsible for RHS. In the present study, we report the clinical features and the molecular investigation of RHS in three unrelated consanguineous Tunisian families including 7 patients with confirmed biochemical diagnosis of tyrosinemia type II. Mutation analyses were performed and two novel missense mutations were identified (C151Y) and (L273P) within exon 5 and exon 8, respectively. The 3D-structural characterization of these mutations provides evidence of defective folding of the mutant proteins, and likely alteration of the enzymatic activity. Phenotype variability was observed even among individuals sharing the same pathogenic mutation.
Mol Genet Metab 2006 Jun
PMID:Clinical and mutational investigations of tyrosinemia type II in Northern Tunisia: identification and structural characterization of two novel TAT mutations. 1657 53


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