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)

Oxidative desulfuration of diethyldithiocarbamate methyl ester (DDTC-Me), a thione xenobiotic and a metabolite of disulfiram, was studied. Using a rat liver microsomal incubation system, DDTC-Me was oxidized at the thionosulfur group, forming DDTC-Me sulfine. Only minimal desulfuration of DDTC-Me to S-methyl-N,N-diethylthiolcarbamate (DETC-Me) occurred. Desulfuration of DDTC-Me increased 4-fold when the microsomal incubation was supplemented with reduced glutathione (GSH) and increased 8-fold when both GSH and glutathione-S-transferase (EC 2.5.1.18) were added. Similar results were obtained using a simplified system containing DDTC-Me sulfine, GSH, and glutathione-S-transferase. This suggested that DDTC-Me sulfine is a stable intermediate formed before DDTC-Me is desulfurated to DETC-Me. This unprecedented desulfuration process can be explained as follows. GSH attacks the oxithiirane isomer of DDTC-Me sulfine, resulting in ring opening followed by loss of glutathione hydrodisulfide, which is reduced by GSH to oxidized glutathione and H2S. GSH can also reduce DDTC-Me sulfine to DDTC-Me. This mechanism is supported by in vitro studies. An approximately 1:1 stoichiometry was observed for the formation of H2S and DETC-Me. A 1:1 stoichiometry was also observed for the consumption of DDTC-Me sulfine, formation of DETC-Me plus DDTC-Me, and formation of oxidized glutathione. Glutathione hydrodisulfide was trapped by derivatization in situ using 4-vinylpyridine. Oxidative desulfuration of a series of dithiocarbamate esters also followed a similar mechanism.
Mol Pharmacol 1994 Dec
PMID:Glutathione- and glutathione-S-transferase-dependent oxidative desulfuration of the thione xenobiotic diethyldithiocarbamate methyl ester. 780 45

The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.
Mol Cell Biol 1995 Feb
PMID:Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains. 782 43

Screening of a mouse liver lambda gt 11 cDNA library with a rat liver 11 beta-hydroxysteroid dehydrogenase cDNA (11 beta-HSDr1A) and subsequent screening with an isolated mouse probe, resulted in the isolation and structure determination of a mouse cDNA encoding an amino acid sequence which is very similar to human and rat 11 beta-hydroxysteroid dehydrogenases (78% and 86% similar, respectively), and also to other known vertebrate 11 beta-hydroxysteroid dehydrogenase structures. Open-reading-frame analysis and the deduced amino acid sequence predict a protein with a molecular mass of 32.3 kDa which belongs to the superfamily of the short-chain dehydrogenase proteins. The amino acid sequence contains two potential glycosylation sites. These data are in agreement with information on the glycoprotein character of the native enzyme. This kind of post-translational modification seems to be a determining factor concerning the equilibrium of the catalyzed 11 beta-dehydrogenation/11-oxo reduction step [Obeid, J., Curnow, K. M., Aisenberg, J. & White, P.C. (1993) Mol. Endocrinol. 7, 154-160; Agarwal, A.K., Tusie-Luna, M.T., Monder, C. & White, P.C. (1990) Mol. Endocrinol. 4, 1827-1832]. After in vitro transcription/translation of the mouse cDNA, immunoprecipitation with anti-(microsomal carbonyl reductase) serum and N-terminal sequence analysis of the purified protein confirms the identity of microsomal 11 beta-hydroxysteroid dehydrogenase with the previously described, microsomal-bound xenobiotic carbonyl reductase [Maser, E. & Bannenberg, G. (1994) Biochem. Pharmacol. 47, 1805-1812], and points to an involvement of the 11 beta-HSD1A isoform in the reductive phase-I metabolism of xenobiotic compounds, besides its endocrinological functions. The alignment and comparison to other hydroxysteroid dehydrogenase forms of the same protein superfamily allows the identification of important residues in the 11 beta-HSD primary structure.
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PMID:Cloning and primary structure of murine 11 beta-hydroxysteroid dehydrogenase/microsomal carbonyl reductase. 785 87

Glutathione S-transferase (GST), an essential detoxification enzyme in parasitic helminths, is a major vaccine target and an attractive drug target against schistosomiasis and other helminthic diseases. Crystal structures of the 26 kDa GST from the helminth Schistosoma japonica (SjGST) have been determined for the unligated enzyme (resolution = 2.4 A, R-factor = 19.7%) and for the enzyme bound to the leading antischistosomal drug praziquantel (resolution = 2.6 A, R-factor = 21.2%). The protein, recombinantly expressed using the Pharamacia PGEX-3X vector for production of GST fusion proteins, contains all 218 residues of SjGST and an additional 13 residues at the C terminus. The structure of unligated SjGST shows that the glutathione binding site pre-exists unchanged in the ligand-free enzyme and is conserved between parasitic and the mammalian class mu enzymes. At therapeutic concentrations the leading antischistosomal drug praziquantel (PZQ) binds one drug per enzyme homodimer in the dimer interface groove adjoining the two catalytic sites. This establishes a protein target for PZQ, identifies the GST non-substrate ligand transport site, and implicates PZQ in steric inhibition of SjGST catalytic and transport for large ligands. Thus, increased expression or mutagenesis of SjGST by the parasite may confer resistance to PZQ. Differences in the xenobiotic binding region between parasitic and mammalian GSTs reveal a distinct substrate repertoire for SjGST and, together with the newly identified PZQ binding site, provide the basis for design of novel antischistosomal drugs. Due to the widespread use expression systems based on SjGST fusions, the atomic structure of SjGST should also provide an important tool for phasing fusion protein structures by molecular replacement.
J Mol Biol 1995 Feb 10
PMID:Crystal structures of a schistosomal drug and vaccine target: glutathione S-transferase from Schistosoma japonica and its complex with the leading antischistosomal drug praziquantel. 785 99

Male Fischer rats were maintained for a period of 17 weeks on an iron-deficient diet along with suitable controls. The effect of long term deprivation of iron on xenobiotic metabolism was studied by the activities of various drug metabolising enzymes in both liver as well as extra-hepatic tissues like lungs, kidneys and intestinal mucosa (I.M.). The results show that among the Phase I (activating) enzymes, the hepatic activities of benzo(a)pyrene hydroxylase (AHH) and microsomal epoxide hydrolase (mEH) are significantly reduced in iron deficiency. The other parameters of the activating system, namely cytochrome P450, aminopyrene demethylase (ADM) and aniline hydroxylase (AH), are not altered. Of the two Phase II (conjugating) enzymes studied, only uridine diphospho glucuronyl transferase (UDPGT) is found to be depressed, but not glutathione S-transferase (GST) in liver in iron deficiency. Activities of Phase I enzymes are markedly lowered in extra-hepatic tissues compared to liver; such depression is not observed in conjugating enzymes. Iron deficiency does not seem to make much impact on the enzyme activities of extra-hepatic tissues. Overall, the hepatic results suggest a defect in detoxification mechanisms in iron deficiency. Such impairment may very well predispose an iron-deficient host to an increased risk of carcinogenesis.
Comp Biochem Physiol B Biochem Mol Biol 1995 Jan
PMID:Effect of long term iron deficiency on the activities of hepatic and extra-hepatic drug metabolising enzymes in Fischer rats. 785 40

Activation of arylamines to mutagenic metabolites by hepatic S9 fractions has been evaluated as a biomaker of fish exposure to pollutants, using gilthead seabream (Sparus aurata), a valuable fish species from the Spanish South Atlantic littoral, as model organism. To obtain maximal sensitivity to the mutagenic action of aromatic amines, a strain of Salmonella typhimurium overproducing O-acetyltransferase was used. Fish were treated with Aroclor 1254, pesticides (malathion and dieldrin), or copper(II), and compared to Aroclor 1254-treated rats. The promutagen activation capabilities of the S9 fractions were further characterized by studying the effect of two monooxygenase inhibitors, alpha-naphthoflavone, a well known inhibitor of aromatic hydrocarbon-inducible forms of cytochrome P450, and methimazole, a substrate for the flavin monooxygenase (FMO) system. This study shows that 2-aminoanthracene (2-AA) and 2-acetylaminofluorene (AAF) activation by gilthead liver is enhanced by treatment of fish with different xenobiotics. The catalyst responsible for this enhanced activation appears to be different for each promutagen and, at least for 2-AA, dependent on the type of xenobiotic. The data presented indicate further that treatment of gilthead with some compounds, such as malathion and dieldrin, enhances the activation of aromatic amines in liver, without inducing ethoxyresorufin-O-deethylase activity. The use of acetyltransferase-overproducing bacteria appears to be a useful tool in the study of arylamine activation by fish liver, where biotransformation capability is lower than in mammals.
Environ Mol Mutagen 1995
PMID:Metabolic activation of carcinogenic aromatic amines by fish exposed to environmental pollutants. 787 26

Expression of functional cytochrome P450 (CYP) isoforms in human embryonic tissues was explored during organogenesis (days 50-60 of gestation) with substrate probes, inhibitor probes, and immunoprobes and by reverse transcription-polymerase chain reaction (PCR), cloning, and sequencing. Evidence was obtained for the presence of relatively high levels of one or more functional CYP3A isoforms in embryonic livers. This was manifested as relatively extensive hydroxylation of (R)-warfarin at carbon 10 and as triacetyloleandomycin-inhibited O-debenzylation of benzyloxyresorufin when human embryonic hepatic microsomal fractions were used as enzyme sources. Immunoblots with anti-CYP3A4 antibody exhibited a strong signal in embryonic hepatic tissues but, in contrast, indicated very low or negligible CYP3A levels in human embryonic lung, kidney, heart, adrenal, and brain tissues. To explore expression of individual members of the CYP3A subfamily in human embryonic hepatic tissues at this early gestational stage, CYP3A cDNA was generated by reverse transcription, amplified by PCR, cloned, and sequenced. Oligonucleotide primers used for PCR were designed to flank target sequences unique to CYP3A but also common to all human CYP3A subfamily members for which GenBank nucleotide sequence information was available (CYP3A3, CYP3A4, CYP3A5, CYP3A5P, and CYP3A7). Sequencing data indicated that plasmids in 58 of 59 recombinant positive colonies contained an insert with a sequence identical to that present in CYP3A7 cDNA and the plasmid of only one colony contained an insert with a sequence identical to that present in CYP3A5 cDNA. No evidence was found for expression of CYP3A3 or CYP3A4. Thus, during organogenesis, human embryonic hepatic tissues express primarily CYP3A7 and are capable of significant CYP3A7-catalyzed xenobiotic monooxygenation during this very early stage of gestation.
Mol Pharmacol 1994 Nov
PMID:Functional cytochrome P4503A isoforms in human embryonic tissues: expression during organogenesis. 796 81

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.
Mol Cell Biol 1994 Dec
PMID:Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation. 796 69

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.
Mol Cell Biol 1994 Sep
PMID:Down-regulation of nuclear aryl hydrocarbon receptor DNA-binding and transactivation functions: requirement for a labile or inducible factor. 806 2

The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT.
Mol Cell Biol 1994 Sep
PMID:Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT). 806 41


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