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)

Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding sites. Initial results showed that oxidation of MDZ by CYP3A4 causes time- and concentration-dependent enzyme inactivation with K(I) and k(inact) values of 5.8 microM and 0.15 min(-1), respectively. The different time courses of MDZ hydroxylation by mutants that predominantly formed 1'-OH MDZ as opposed to 4-OH MDZ provided strong evidence that the 1'-OH MDZ pathway leads to CYP3A4 inactivation. Correlational analysis of 1'-OH formation versus 4-OH formation by the mutants supports the inference that the two metabolites result from the binding of MDZ at two separate sites. Thus, substitution of residues Phe-108, Ile-120, Ile-301, Phe-304, and Thr-309 with a larger amino acid caused an increase in the ratio of 1'-OH/4-OH MDZ formation, whereas substitution of residues Ser-119, Ile-120, Leu-210, Phe-304, Ala-305, Tyr-307, and Thr-309 with a smaller amino acid decreased this ratio. Kinetic analyses of nine key mutants revealed that the alteration in regioselectivity is caused by a change in kinetic parameters (V(max) and K(M)) for the formation of both metabolites in most cases. The study revealed the role of various active-site residues in the regioselectivity of MDZ oxidation, identified the metabolic pathway that leads to enzyme inactivation, and provided an indication that the two proposed MDZ binding sites in CYP3A4 may be partially overlapping.
Mol Pharmacol 2002 Mar
PMID:Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation. 1185 29

Cytochromes P450 (P450s) are hemoprotein enzymes committed to the metabolism of chemically diverse endo- and xenobiotics. They are anchored to the endoplasmic reticulum (ER) membrane with the bulk of their catalytic domain exposed to the cytosol, and thus they constitute excellent examples of integral monotopic ER proteins. Physiologically they are known to turn over asynchronously, but the determinants that trigger their proteolytic disposal and the pathways for such cellular disposal are not well defined. We recently showed that CYP3A4, the dominant human liver drug-metabolizing enzyme, and its rat liver orthologs undergo ubiquitin-dependent 26S proteasomal degradation not only after suicide inactivation, but also when CYP3A4 is expressed in Saccharomyces cerevisiae, presumably in its "native" form. The latter findings, obtained by the use of strains either with compromised proteasomal degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) or deficient in ubiquitin-conjugating enzymes (Ubc; UBC), revealed that this native monotopic P450 enzyme, in common with the polytopic HMGR, required the function of certain HRD (HMGR degradation) and UBC genes. In this study, we examined the degradation of CYP2C11, a male rat liver-specific P450, by heterologous expression in S. cerevisiae under comparable conditions. We report that unlike CYP3A4 and HMGR, the degradation of CYP2C11 in S. cerevisiae is independent of either HRD or UBC gene function, but it is largely dependent on vacuolar (lysosomal) proteolysis. These findings with two monotopic ER hemoproteins, CYP2C11 and CYP3A4, and the polytopic ER protein HMGR attest to the remarkable mechanistic diversity of cellular proteolytic disposal of ER proteins.
Mol Pharmacol 2002 May
PMID:Native CYP2C11: heterologous expression in Saccharomyces cerevisiae reveals a role for vacuolar proteases rather than the proteasome system in the degradation of this endoplasmic reticulum protein. 1196 Nov 33

We recently demonstrated that a variant allele of CYP3A5 (CYP3A5*3) confers low CYP3A5 expression as a result of improper mRNA splicing. In this study, we further evaluated the regulation of CYP3A5 in liver and jejunal mucosa from white donors. For all tissues, high levels of CYP3A5 protein were strongly concordant with the presence of a wild-type allele of the CYP3A5 gene (CYP3A5*1). CYP3A5 represented greater than 50% of total CYP3A content in nearly all of the livers and jejuna that carried the CYP3A5*1 wild-type allele. Overall, CYP3A5 protein content accounted for 31% of the variability in hepatic midazolam hydroxylation activity. Improperly spliced mRNA (SV1-CYP3A5) was found only in tissues containing a CYP3A5*3 allele. Properly spliced CYP3A5 mRNA (wt-CYP3A5) was detected in all tissues, but the median wt-CYP3A5 mRNA was 4-fold higher in CYP3A5*1/*3 livers compared with CYP3A5*3/*3 livers. Differences in wt-CYP3A5 and CYP3A4 mRNA content explained 53 and 51% of the interliver variability in CYP3A5 and CYP3A4 content, respectively. Hepatic CYP3A4 and CYP3A5 contents were not correlated when all livers were compared. However, for CYP3A5*1/*3 livers, levels of the two proteins were strongly correlated (r = 0.93) as were wt-CYP3A5 and CYP3A4 mRNA (r = 0.76). These findings suggest that CYP3A4 and CYP3A5 genes share a common regulatory pathway for constitutive expression, possibly involving conserved elements in the 5'-flanking region.
Mol Pharmacol 2002 Jul
PMID:Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. 1206 67

We report the development of a rapid real-time assay that measures the transcription of luciferase reporter genes in transduced mouse hepatic cells in vivo. Luciferase activity is noninvasively measured by whole-body optical imaging within hours of the hydrodynamic injection of as little as 1 microg of naked DNA. Transcription of genes introduced as linearized DNA can be serially assayed for weeks in each animal. Transcription was quantified by extracorporal monitoring of bioluminescence as well as or better than by traditional in vitro bioluminescence assay. Our assay allows the measurement of transcription as it occurs, under the most informative biological conditions (i.e., in a living, intact organ). Furthermore, it substantially reduces the cost, time, and number of animals required for analysis of gene expression. The utility of the method is demonstrated in the discovery that topotecan and etoposide are ligands of pregnane X receptor that induce CYP3A4 transcription.
Mol Pharmacol 2002 Sep
PMID:Development of a real-time in vivo transcription assay: application reveals pregnane X receptor-mediated induction of CYP3A4 by cancer chemotherapeutic agents. 1218 18

The results of homology modelling of the human glucorticoid receptor (hGR) ligand-binding domain (LBD) based on the ligand-bound domain of the human estrogen receptor alpha (hERalpha) are reported. It is shown that known hGR ligands which induce the human cytochrome P450 enzyme CYP3A4 are able to fit the putative ligand-binding site of the nuclear hormone receptor and form hydrogen bonds with key amino acid residues within the binding pocket. Quantitative structure-activity relationships (QSARs) have been derived for hGR-mediated CYP3A4 induction which involve certain molecular structural and physicochemical properties of the ligand themselves, yielding good correlations (R=0.96-0.98) with fold induction of CYP3A4 known to be mediated via hGR involvement.
J Steroid Biochem Mol Biol 2002 Oct
PMID:Molecular modelling of the human glucocorticoid receptor (hGR) ligand-binding domain (LBD) by homology with the human estrogen receptor alpha (hERalpha) LBD: quantitative structure-activity relationships within a series of CYP3A4 inducers where induction is mediated via hGR involvement. 1247 85

The steroid and xenobiotic receptor (SXR) is an orphan nuclear receptor that plays a key role in the regulation of xenobiotic response by controlling the expression of drug metabolizing and clearance enzymes. We observed that pregnane X receptor (PXR), the mouse ortholog of SXR, was retained in the cytoplasm of hepatic cells of untreated mice, whereas PXR was translocated to the nucleus after administration of a ligand, pregnenolone 16 alpha-carbonitrile. To understand the molecular mechanisms underlying the xenochemical-dependent nuclear translocation of SXR, we identified the signal sequence of SXR that regulates its nuclear translocation; using an in vitro expression system, we allocated the nuclear localization signal (NLS) to amino acid residues 66 to 92 within the DNA binding domain of SXR. The NLS of SXR is characterized as the bipartite type, and is recognized by the three molecular species of importin alpha: Rch1 (PTAC58), NPI1, and Qip1, in the presence of PTAC97 of importin beta to target the nuclear pore. The nuclear translocation of SXR was observed as an essential regulatory event for transcription of its target genes such as CYP3A4. These results strongly suggest that the molecular mechanism of the nuclear import of SXR was different from that of another xenosensor, the constitutively active receptor, whose translocation into the nucleus is mediated by a leucine-rich xenochemical response signal in its ligand binding domain.
Mol Pharmacol 2003 Mar
PMID:Molecular mechanism of nuclear translocation of an orphan nuclear receptor, SXR. 1260 58

The liver is an essential organ in humans not only for the production and storage of energy but also for detoxification of chemical compounds, but knowledge about changes in the gene expression profile in the human liver during the prenatal and postnatal periods is limited. Profiling of genes differentially expressed between the fetal liver (FL) and the postnatal liver (PNL) is one of the methods to investigate candidates affecting the difference in biological characteristics between FL and PNL. To identify genes differentially expressed between FL and PNL (childhood and adult liver), we analyzed the gene expression profiles across 9 FL and 14 PNL samples using a high-density oligonucleotide DNA array. Using Mann-Whitney U test followed by k-nearest-neighbors (supervised learning method) and hierarchical clustering (unsupervised learning method) algorithms, we found 33 genes clearly discriminating between the FL group and PNL group. The functional classification of the 33 genes identified was related to several kinds of biological pathways, regulating the cell cycle (PCNA, CDC7L1, CCND3, YWHA1, PKMYT1), DNA replication and repair (RFC4, RECQ2, PCNA, NAP1L1), cell growth (IGF2, IGFBP2, PRSS11), hormonal signals (AR, SRD5A1, NR1I3), and cellular metabolism (E2-EPF, WWP1, CYP2C9, CYP2E1, CYP2A6, CYP2A7, CYP2A13, CYP4F2, CYP3A4, DDT). The results presented herein provide evidence of a differential expression profile of genes regulating the cell cycle, DNA replication and repair, cell growth, regulation of hormonal signals, and cellular metabolism, between FL and PNL in humans. The 33 genes identified in this study are suggested to be useful markers clearly discriminating between FL and PNL using the gene expression profile.
Int J Mol Med 2003 Jun
PMID:Profiling of genes differentially expressed between fetal liver and postnatal liver using high-density oligonucleotide DNA array. 1273 11

CYP3A4, the predominant but variably expressed cytochrome P450 of adult human liver, is subject to multifaceted constitutive regulation as well as transcriptional induction by a variety of structurally unrelated xenobiotics. Using transient transfections in HepG2 cells, we previously demonstrated the existence of a potent xenobiotic-responsive enhancer module located between - 7.2 and - 7.8 kilobases upstream of the CYP3A4 transcription start site. Induction is mediated by interaction of transcription factor binding sites in the XREM with the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). To determine the in vivo relevance of these findings and to establish a mouse model of human CYP3A4 regulation, we have generated transgenic mice carrying constructs comprising the upstream regulatory region of the human CYP3A4 gene linked to the lacZ reporter gene. Constitutive expression was observed in a developmental, tissue- and cell-specific fashion that mirrors the human situation. In addition, robust hepatic and intestinal induction with a range of reagents known to activate PXR and/or CAR (e.g., dexamethasone, pregnenolone 16alpha-carbonitrile, and phenobarbital) was observed. However, no expression or induction was apparent with a construct lacking upstream sequences beyond - 3.2 kilobases. Histochemical staining for beta-galactosidase activity revealed that dose-dependent increases in transgene levels were associated with a zonal expansion of lacZ expressing hepatocytes, suggesting that xenobiotic induction of CYP3A genes operates primarily through the recruitment of more cells committed to expression. In summary, CYP3A4/lacZ transgenic mice provide an in vivo model for the study of the molecular mechanisms involved in the regulation of a significant human drug metabolizing enzyme.
Mol Pharmacol 2003 Jul
PMID:Transgenic mouse models of human CYP3A4 gene regulation. 1281 59

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

All-trans-retinoic acid (ATRA) is used in the treatment of promyelocytic acute leukemia. The biotransformation of this drug is catalyzed by various cytochrome P450 (CYP) enzymes, but relatively little is known about the effect of ATRA on CYP enzyme expression in leukemic cells. In the present study, we conducted transcript profiling of CYP and related genes in cultured HL-60 human promyelocytic leukemic cells and determined the effect of ATRA on the expression of these genes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with a block-cycler indicated the presence of CYP1B1 but not CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP3A4, CYP3A5, or CYP26A1 transcript in cultured HL-60 cells. ATRA treatment (0.1-40 microM for 3 days) increased CYP1B1 mRNA levels by up to 3 fold, as determined by a quantitative real-time PCR method. The same ATRA treatment also resulted in the detection of CYP26A1 but not CYP1A1, CYP2B6, CYP2C8, CY2C9, CYP3A4, or CYP3A5 mRNA. Additional experiments showed that phenobarbital increased CYP2B6 mRNA expression and that pregnane X receptor (PXR) but not constitutive androstane receptor (CAR) was detected in HL-60 cells. Overall, our novel findings indicate the upregulation of CYP1B1 by ATRA in HL-60 human promyelocytic leukemic cells shown for the first time to express PXR but not CAR mRNA.
Mol Cell Biochem 2003 Jun
PMID:Transcript profiling of cytochrome P450 genes in HL-60 human leukemic cells: upregulation of CYP1B1 by all-trans-retinoic acid. 1287 Jun 55


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