Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The function of the hormone-binding domain of the human progesterone receptor was examined in a yeast cell system and in mammalian HeLa cells using mutant receptors, progesterone, the progesterone agonist R502, and the antagonists RU-486, Org31806, and Org31376. The hormone-binding domain, located on the carboxy terminal of the peptide, is known to initiate a conformational change in the receptor upon binding an agonist or antagonist, then shedding of associated proteins including the heat shock protein, dimerization of the receptor, and finally, binding to DNA, leading to transcription. Binding of a progesterone antagonist such as RU-486 elicits all these events except transcription. First the progesterone receptor was inserted in yeast with a plasmid, and a set of mutants were generated, using beta galactosidase as an indicator. A mutant progesterone receptor, U-P1, was selected for mechanistic studies, that binds and was activated by antagonists, but was inactive with progesterone. This receptor had a deletion at base 2636, resulting in a shift of reading frame so that a stop codon 36 nucleotides downstream caused truncation of 54 amino acids at the C-terminus and addition of 12 novel amino acids. Western blot analysis confirmed the expected molecular weight. The mutant receptor was active with RU-486, suggesting that the C-terminus may be responsible for poor transcription with RU-486, suggesting in normal receptors. 2 other truncated mutants were inactive with progesterone. These data suggested that the terminal 42 amino acids of the progesterone receptor are needed to bind progesterone, and that the antagonist is contacting different amino acids than the native receptor, possibly inducing a different conformational change. The activity of the UP-1 mutant was also confirmed in HeLa cells, with the chloramphenicol acetyltransferase reporter system. The results were interpreted to mean that progesterone agonists and antagonists contact at least some different amino acids in the hormone binding domain of the receptor, and that the conformational changes resulting from binding these agents are different. It appears that the C-terminus of the receptor contains an inhibitory domain which, when removed, turns antagonists into agonists.
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PMID:The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor. 158 49

Both cellular activation signals and exposure to glucocorticoids such as dexamethasone (Dex) cause programmed cell death in T cell hybridomas. When cells were activated in the presence of Dex, however, the degree of killing that was achieved by either stimulus alone was markedly reduced. Dex-induced programmed cell death of normal T cell clones was also prevented by cellular activation. Cyclosporin A (CsA) completely blocked the activation-induced death of T cell hybridomas, but actually enhanced the killing caused by Dex. The addition of CsA to activated T cell hybridomas in the presence of Dex allowed killing to proceed, consistent with ability of CsA to block activation-induced nuclear gene transcription. A number of independent approaches were used to explore the effect of activation on the glucocorticoid signaling/effector pathway. First, RU-486, which binds the glucocorticoid receptor and is a potent competitive antagonist of Dex, did not inhibit activation-induced cell killing. Second, activation of T cell hybridomas did not cause the translocation of the glucocorticoid receptor from the cytoplasm to the nucleus, nor did it prevent the receptor translocation induced by treatment with Dex. Finally, T cell hybridomas were transfected with a plasmid containing the chloramphenicol acetyltransferase (CAT) gene under the control of two tandemly arranged glucocorticoid-responsive elements. Activation of these cells did not induce CAT activity, and did not inhibit the CAT activity induced by Dex. In fact, there was a paradoxical increase in CAT activity when cells were treated with both stimuli. We conclude that cellular activation does not directly utilize the glucocorticoid receptor nor the glucocorticoid pathway when inducing programmed cell death. Furthermore, the ability of activation to inhibit Dex-mediated killing is not due to interference with the classical glucocorticoid signaling pathway, up to and including the initiation of gene transcription. Alternative mechanisms of antagonism, as well as the possible relevance of this phenomenon to the positive selection of self-recognizing thymocytes, are discussed.
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PMID:Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic. 197 85

The brain isozyme of creatine kinase (CKB) is a major component of the estrogen-induced proteins in the rat uterus. Hormonal specificity of this response was studied in cotransfection assays using the rat CKB promoter linked to the bacterial chloramphenicol acetyltransferase gene. Response was specific for estrogen as 17 beta-estradiol in the presence of estrogen receptor dramatically stimulated the CKB promoter. This induction was completely blocked by the estrogen antagonist ICI 164,384. Nuclear receptors for progesterone, androgen, glucocorticoid and vitamin D did not significantly activate the CKB promoter in the presence of their respective ligands. Creatine kinase (CK) activity was analyzed in decidualized mouse uterus to assess estrogenic activity in vivo. Upon oil stimulation, uterine horns of day 4 pseudopregnant mice underwent a dramatic outgrowth in response to endogenous progesterone. This response was accompanied by a significant decrease in CK activity from a control value of 1.44 +/- 0.25 to 0.38 +/- 0.08 IU/mg protein (P < 0.001), indicating that the action of estrogen was suppressed. Treatment of females one day prior to oil-stimulation with progesterone receptor antagonists, RU486 (Mifepristone) or ZK299 (Onapristone), or with a monoclonal antibody to progesterone (DB3), abolished decidualization, and also restored the CK activity to the control value. These results suggest that CK can be used as a specific cellular marker to detect unopposed estrogen action in the mouse uterus associated with progesterone withdrawal or receptor blockade.
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PMID:Creatine kinase activity as an indicator of unopposed estrogen action in the mouse uterus associated with anti-progesterone treatment. 803 8

The human liver cytochromes P450 3A (CYP3As), orthologous to the rat glucocorticoid inducible forms, are composed of at least four differentially expressed members. To begin the study of the molecular events in the glucocorticoid regulation of CYP3A5, we fused 5' sequences of CYP3A5 to the chloramphenicol acetyltransferase gene in a vector that contains the herpes simplex virus thymidine kinase promoter. In HepG2 cells, the largest 5' CYP3A5 gene fragment (1.4 kb) suppressed the TK promoter. However, suppression was overcome by addition of 10 microM dexamethasone. A series of unidirectional deletions revealed a unique 219-bp fragment (-891 to -1109 bp upstream from the transcriptional start site) that conferred dexamethasone responsiveness on the TK promoter regardless of either the distance or orientation from the promoter and thus appears to be an enhancer. Nucleotide sequence analysis of this CYP3A5 enhancer revealed no consensus 15-bp glucocorticoid responsive element (GRE) (GGTACANNNTGTTCT); however, two GRE "half-sites" (TGTTCT) were found separated by 160 bp. Although dexamethasone stimulated the CYP3A5 enhancer only 3-4-fold in HepG2 cells, the CYP3A5 enhancer was stimulated 7- and 12-fold in immortalized primary human hepatocytes and primary rat hepatocyte cultures, respectively. The glucocorticoid receptor (GCR) seems to be indispensable to this process because 1) dexamethasone induction can be blocked by the antiglucocorticoid RU-486, 2) dexamethasone-dependent transcriptional activation of the CYP3A5 enhancer in HepG2 cells required cotransfection of an expression vector containing the intact GCR, yet 3) cotransfection with a plasmid that contains a mutation in the ligand binding domain of the GCR does not activate the CYP3A5 enhancer in the presence of dexamethasone. To further localize the dexamethasone responsive region of the 219-bp CYP3A5 enhancer, it was subdivided and fused to the TKCAT expression vector. Transfection analysis in HepG2 cells demonstrated that neither GRE half-site can independently confer dexamethasone responsiveness on the TK promoter. Block mutations of either of the two GRE half-sites or point mutations at specific GCR binding sites eliminates dexamethasone inducibility, demonstrating the half-sites need to interact. Electromobility shift assays indicate that the CYP3A5 5'-GRE half-site 1) specifically binds purified GCR, 2) can displace binding of the GCR to a consensus GRE, and 3) shifts a protein in HepG2 nuclear extracts that is supershifted by GCR antibody, demonstrating that this enhancer is an authentic GRE. This is the first study to demonstrate that a member of the human CYP3A gene family contains an enhancer that binds the GCR and that this binding is critical to transcriptional activation by dexamethasone.
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PMID:Identification of a novel dexamethasone responsive enhancer in the human CYP3A5 gene and its activation in human and rat liver cells. 856 13