NADPH-cytochrome P-450 Oxidoreductase Gene Research Articles

Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase.

The diurnal variation in acetaminophen (APAP) hepatotoxicity (chronotoxicity) reportedly is driven by oscillations in metabolism that are influenced by the circadian phases of feeding and fasting. To determine the relative contributions of the central clock and the hepatocyte circadian clock in modulating the chronotoxicity of APAP, we used a conditional null allele of brain and muscle Arnt-like 1 (Bmal1, aka Mop3 or Arntl) allowing deletion of the clock from hepatocytes while keeping the central and other peripheral clocks (e.g., the clocks controlling food intake) intact. We show that deletion of the hepatocyte clock dramatically reduces APAP bioactivation and toxicity in vivo and in vitro because of a reduction in NADPH-cytochrome P450 oxidoreductase gene expression, protein, and activity.

Downregulation of Mouse Hepatic CYP3A Protein by 3-Methylcholanthrene Does Not Require Cytochrome P450-Dependent Metabolism

The aryl hydrocarbon receptor (AHR)-dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons is well characterized. We reported previously that MC treatment triggers a pronounced downregulation, particularly at the protein level, of mouse hepatic Cyp3a11, a counterpart of the key human drug-metabolizing enzyme CYP3A4. To determine whether this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of the NADPH-cytochrome P450 oxidoreductase gene. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein immunoreactivity were elevated by approximately 9-fold in LCN mice compared with wild-type (WT) mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein immunoreactivity, and CYP3A catalytic activity in WT mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by MC was suggested previously to occur via an AHR-dependent mechanism requiring conversion of the parent compound to DNA-damaging reactive metabolites; however, hepatic FMO3 mRNA levels were dramatically increased by MC in both WT and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated WT mice, under conditions in which 1-aminobenzotriazole caused marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity. These results indicate that MC downregulates mouse hepatic CYP3A protein via a pretranslational mechanism that does not require hepatic microsomal P450-dependent activity.

Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus.

Resistance to antifungal drugs, specifically azoles such as fluconazole, in the opportunistic yeast Candida albicans has become an increasing problem in human immunodeficiency virus (HIV)-infected individuals. The molecular mechanisms responsible for this resistance have only recently become apparent and can include alterations in the target enzyme of the azole drugs (lanosterol 14alpha demethylase [14DM]), or in various efflux pumps from both the ABC transporter and major facilitator gene families. To determine which of these possible mechanisms was associated with the development of drug resistance in a particular case, mRNA levels have been studied in a series of 17 clinical isolates taken from a single HIV-infected patient over 2 years, during which time the levels of fluconazole resistance of the strain increased over 200-fold. Using Northern blot analysis of steady-state levels of total RNA from these isolates, we observed increased mRNA levels of ERG16 (the 14DM-encoding gene), CDR1 (an ABC transporter), and MDR1 (a major facilitator) in this series. The timing of the increase in mRNA levels of each of these genes correlated with increases in fluconazole resistance of the isolates. Increased mRNA levels were not observed for three other ABC transporters, two other genes in the ergosterol biosynthetic pathway, or the NADPH-cytochrome P-450 oxidoreductase gene that transfers electrons from NADPH to 14DM. Increases in mRNA levels of ERG16 and CDR1 correlated with increased cross-resistance to ketoconazole and itraconazole but not to amphotericin B. A compilation of the genetic alterations identified in this series suggests that resistance develops gradually and is the sum of several different changes, all of which contribute to the final resistant phenotype.

Cloning and expression in Escherichia coli and Saccharomyces cerevisiae of a novel tobacco cytochrome P-450-like cDNA

A cDNA library constructed from poly(A) + RNA of tobacco BY2 cells treated with 2,4-dichlorophenoxyacetic acid was screened by using a synthetic oligonucleotide corresponding to the heme binding region of avocado CYP71A1. A cloned 2-kb cDNA designated as cTBP contained an open reading frame of 1593 by encoding a protein of molecular size of 58 916. The deduced amino acid sequence included a cysteine residue corresponding to fifth ligand of heme-Fe at 497th. The coding sequence was expressed under the control of tac promoter and rrnB terminator in Escherichia coli to yield 7 to 10 nmol P450 equivalent per litre of the culture in the presence of δ-aminolevulinic acid. The modified coding sequences in which NH2-terminal residues 2–25 were replaced by the NH2-terminal 18 amino acid residues of microsomal bovine CYP17 were also expressed under the control of ADH promoter and terminator in Saccharomyces cerevisiae to yield 29 and 30 pmol of P450 equivalent/mg protein in the microsomal fraction, respectively. On co-expression of each of the modified coding sequences and yeast NADPH-cytochrome P-450 oxidoreductase gene, the yeast microsomas exhibited 7-ethoxycoumarin O-deethylase activity. Based on these results, tobacco cTBP was found to encode a novel P450-like species with a monooxygenese activity related to xenobiotic metabolism.

Transcriptional Regulation of the TATA-less NADPH Cytochrome P-450 Oxidoreductase Gene

Multiplecis-acting DNA sequences regulating expression of the rat liver NADPH-cytochrome P-450 oxidoreductase gene have been identified in transient transfection assays using promoter deletion constructs linked to the chloramphenicol acetyl transferase gene. The TATA-less promoter possesses nine GC-boxes which contain the consensus sequence for the transcription factor Sp1. While loss of the seven distal GC-boxes had minimal effect on transcriptional activity, deletion of the next 35 bp, from −206 to −172, resulted in ∼90% loss of promoter activity. Contained within this region is an Sp1 binding site indicating that either (1) this particular consensus sequence was essential for transcription, (2) the two proximal GC boxes act in concert, or (3) a yet unidentified regulatory element resides within this 35-bp stretch. In addition, transfection experiments demonstrated that two separate distal regions (−622 to −1167 and −1500 to −2300) contain negative regulatory elements which down-regulate gene transcription in a position-independent manner. Mobility-shift analyses and DNase footprinting identified sequences in the proximal region of the promoter that bound proteins present in nuclear extracts. Four protected segments were observed within the first 100 bp upstream of the transcription start site; these include (1) the region encompassing the transcription start site (−7 to +4), (2) the region normally occupied by a TATA-box (−38 to −18), (3) the bases from −78 to −60 which contain the regulatory element CACC, and (4) bases −105 to −92 which include an Sp1 binding site. Hence, regulation of the NADPH-cytochrome P-450 oxidoreductase gene is controlled by both positive and negative regulatory elements, and, of the nine Spl consensus sites, the two proximal sites are sufficient to support basal transcription.

Cloning and characterization of the NADPH cytochrome P450 oxidoreductase gene from the filamentous fungus Aspergillus niger.

In this paper, we describe the cloning and molecular characterization of the Aspergillus niger cytochrome P450 reductase (CPR) gene, cprA. Attempts to clone the cprA gene by heterologous hybridization techniques were unsuccessful. Using the polymerase chain reaction (PCR) with degenerate primers based on conserved regions found in cpr genes from other organisms, we were able to isolate a fragment that contained part of the gene. With the aid of this fragment, a genomic fragment containing the entire coding region and 5' and 3' untranslated ends of the cprA gene was isolated and sequenced. The cprA gene was introduced in multiple copies in A. niger strain N402 using the amdS transformation system. One of the resulting transformants, AB2-2, showed a 14-fold increase in CPR activity, indicating that the cloned cprA gene is functional. We analyzed the induction of cprA gene expression by several generally used cytochrome P450 inducers but did not find any induction of cprA gene expression. However, A. niger cprA gene expression could be induced by benzoic acid, which is the substrate of the highly inducible A. niger cytochrome P450 gene, bphA (cyp53). On the basis of a comparison of the deduced protein sequence of the A. niger cprA gene with CPR proteins isolated from other organisms, the structure-function relationship of some conserved regions is discussed.

NADPH Cytochrome P-450 Oxidoreductase Gene: Identification and Characterization of the Promoter Region

The untranslated first exon and the 5′-flanking region for the rat liver NADPH-cytochrome P450 oxidoreductase gene has been isolated from a Wistar-Furth genomic library. The remainder of the gene is composed of 15 exons which code for the mature protein and a 3′-nontranslated segment (T. D. Porter et al. Biochemistry, 1990, 29, 9814-9818). The 56-bp first exon resides 30.5 kb upstream from exon two, making the total gene length approximately 50 kb. While the region surrounding the start site (TC A GAGAC) was found to be homologous to a eukaryotic cap signal, the 5′ flanking region Possesses neither a TATA nor a CCAAT box. Instead it contains five GC-rich hexanucleotide consensus sequences for the transcription factor Sp1. These features clearly distinguish it from genes encoding other members of the mixed-function oxidase system, the cytochromes P450. Primer extension analysis and S1 nuclease mapping identified multiple transcriptional start sites. In many respects, the TATA-less oxidoreductase promoter resembles the promoter regions of dihydrofolate reductase and other housekeeping genes. Northern blot analysis demonstrates that this promoter is modulated by phenobarbital and trans-stilbene oxide, known inducers of oxidoreductase.

NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein

cDNA clones to rat liver NADPH-cytochrome P-450 oxidoreductase were used to isolate genomic clones from a Wistar-Furth inbred rat genomic DNA library. Fifteen exons containing the coding region and 3'-nontranslated segment of the P-450 reductase gene were identified, spanning 20 kilobases of DNA contained in 3 lambda-Charon 35 clones. The organization of this single copy gene reveals a general correspondence between exons and structural domains of the protein, with the segment responsible for anchoring the reductase to the microsomal membrane and several segments involved in FMN, FAD, and NADPH binding encoded by discrete exons.

Isolation and characterization of the alkane-inducible NADPH-cytochrome P-450 oxidoreductase gene from Candida tropicalis. Identification of invariant residues within similar amino acid sequences of divergent flavoproteins.

The gene coding for the Candida tropicalis NADPH-cytochrome P-450 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis lambda gt11 expression library and colony hybridization of a C. tropicalis genomic library. The C. tropicalis CPR gene produces a 2.35-kilobase mRNA transcript, levels of which were shown to be increased 16-fold in cells grown on tetradecane relative to cells grown on glucose as the sole carbon source. A 3-kilobase DNA fragment was sequenced, including 554 and 397 base pairs of 5'- and 3'-noncoding sequence, respectively. A single open reading frame of 2040 base pairs was identified and predicts a 76,683-Da polypeptide of 680 amino acid residues. The deduced C. tropicalis CPR amino acid sequence was compared with each of the CPR sequences reported from other organisms and invariant residues were identified. Multiple pairwise alignments of divergent members of protein families, previously recognized for their sequence similarities in their respective binding domains for FMN, FAD, and NADPH, have allowed identification of a subset of these invariant residues. From these analyses we infer the importance of 25 of the 680 amino acid residues.

Functional Expression of the Alkane-Inducible Monooxygenase System of the Yeast: Candida tropicalis IN Saccharomyces cerevisiae

The genes for the alkane-inducible monooxygenase system of the yeast Candida tropicalis, namely a cytochrome P450alk (P450alk) and a NADPH cytochrome P450 oxidoreductase (NCPR) gene, were transferred in Saccharomyces cerevisiae. The P450alk gene was expressed in this host with the help of the yeast alcohol dehydrogenase I (ADHI) promoter and terminator, whereas the NCPR gene could be expressed with its own structural elements. The presence of P450alk in S. cerevisiae microsomal fractions resulted in a new acquired lauric acid terminal hydroxylation activity. Moreover, the same activity, coupled with the appearance of 12-hydroxylauric acid derivatives, could be obtained by the addition of lauric acid to intact cells expressing P450alk. The coordinate expression of the P450alk and NCPR genes in S. cerevisiae elevated the turnover rate of the P450alk monooxygenase activity about 2-fold.

Cloning and characterization of the rat NADPH-cytochrome P-450 oxidoreductase gene.

A rat liver DNA genomic library was prepared using the lambda phage cloning vector Charon 28. Recombinant phage were screened with a cDNA clone (pOR-7) containing sequences complementary to mRNA coding for NADPH-cytochrome P-450 oxidoreductase. This cDNA clone contains the poly(A) addition site and 60% of the mRNA sequence (Gonzalez, F. J., and Kasper, C. B. (1982) J. Biol. Chem. 257, 5962-5968). Four positive phage were identifed and plaque-purified, and their DNA was isolated and subjected to restriction endonuclease mapping. All phage DNA inserts, which ranged from 11 to 16 kilobases, contained several overlapping restriction fragments. The clone with the largest insert (lambda OR-2) was found to contain restriction fragments identical with those of rat DNA when both were subjected to Southern blotting with nick-translated pOR-7 DNA; this finding established the presence in lambda OR-2 of the 3' end (poly(A) addition site) of the oxidoreductase gene. When [32P]cDNA synthesized from enriched oxidoreductase poly(A) RNA was utilized as a probe, additional fragments were identified. The fragment most distal to the 3'-specific fragment was assumed to contain the 5' cap site and was subcloned into pBR322 for further analysis. Restriction mapping and Southern blot analysis further localized the 5' end of the gene to an AvaII fragment of 540 base pairs (bp). Hybridization of this fragment with oxidoreductase mRNA-enriched poly(A) RNA resulted in the arrest of translation of oxidoreductase; this confirmed that it contained an exon region of the oxidoreductase gene. S1 nuclease mapping and DNA sequencing identified to within +/- 1 bp the 5' cap site of the gene which corresponds to an A start. DNA sequencing of the 5'p flanking region revealed no "TATA box" in the vicinity of -25 to -30 bp of the cap site. R-loop analysis of lambda OR-2 revealed the presence of a minimum of seven introns in the 6000-bp oxidoreductase gene and eight exons with a total length of approximately 2600 bp.