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BIOLOGY OF CYTOCHROME P450 REDUCTASE

Discovery and Characterization

Mammalian NADPH-cytochrome P450 reductase (CPR) was first identified by Horecker in 19501 as an NADPH-specific cytochrome c reductase.  Later studies2 showed that this flavoprotein was situated on the endoplasmic reticulum (microsomes); although cytochrome c, a mitochondrial protein, was evidently not its natural redox partner, this name is still used occasionally.  Studies in the 1960's linked CPR to the newly discovered microsomal electron transport chains, cytochromes P450 and b5, involved in drug and steroid hydroxylations. 

 

Reconstitution of Fatty Acid Hydroxylation
(from Lu and Coon, 1968)
3

  Components Activity
(% of Complete)
  Complete 100
  - P450 17
  - phospholipid 9
  - CPR 10
  - NADPH 0
  - O2 10

This was definitively demonstrated by Lu and Coon when reconstitution of a microsomal fatty acid hydroxylase was shown to require cytochrome P450, CPR, and phospholipid for activity.3 

 

The ability of CPR to transfer electrons to cytochrome b5 linked CPR to the fatty acid desaturase and elongase pathways;4 CPR has similarly been shown to be the redox partner for heme oxidase in the heme degradation pathway5 and for squalene monooxygenase and 7-dehydrocholesterol reductase (sterol delta-7 reductase) in the synthesis of sterols6.    

Expression and Regulation

Hormonal regulation of CPR expressionConsistent with its many functions in the cell, CPR is a widely expressed protein, present at some level in all tissues examined.  It is most abundant in the liver, where the cytochrome P450 system is highly expressed.  Unlike the cytochromes P450, however, CPR levels are not readily modulated by "inducers" of the P450 system; typically a strong P450 inducer, such as phenobarbital or pregnenolone-16a-carbonitrile, produces only a 2-3-fold increase in CPR levels.7  Other inducers, such as 3-methylcholanthrene and ethanol, have little or no effect on CPR levels.

CPR expression is regulated at the transcriptional level by the pituitary-thyroid axis, and thyroid hormone (T3) is necessary to maintain CPR expression.8  Hypophysectomy (removal of the pituitary gland) decreases thyroid hormone expression and results in a >50% decrease in hepatic CPR levels.  Steroidogenic tissues, such as the adrenal glands, contain high levels of CPR; expression in these tissues is regulated by adrenocorticotrophic hormone (ACTH).9

Analysis of the CPR Gene

The CPR gene is greater than 50 kb in length and maps to chromosome 6 in the mouse and 7q11.23 (S90469) in man.10  The exon organization of the coding region (exons 2-16) correlates well with the domain structure of the protein.11  The gene has an extended first intron (30 kb) and multiple transcription start sites; the promoter lacks 'TATA box' and 'CCAAT box' elements, consistent with its role as a 'housekeeping' gene (low-level expression in many tissues).  The promoter contains nine copies of an Sp1 binding-element which appear to act as position-independent enhancers, in contrast to other housekeeping genes in which the proximal Sp1 site is critical to transcriptional initiation:  In the reductase gene the first two Sp1 sites are the most important, but distal sites can compensate for their loss.  Two sites are essential to transcription in liver hepatoma cells, one of which contains both an Sp1 site and an Egr-1 site and may be involved in CPR expression in specific tissues and during development, and a second site (ORU) which appears to be unique to the CPR gene.12  Both elements are necessary for gene transcription.  Consistent with its regulation by thyroid hormone, the promoter contains a thyroid response element (TRE) at position -564 which binds thyroid hormone receptors and confers thyroid hormone responsiveness to reporter gene constructs.13  In addition to transcriptional regulation, thyroid hormone also regulates CPR expression through mRNA stabilization, particularly in the normal, euthyroid state.13 

CPR gene diagram

 

References
  1. Horecker BL.  Triphosphopyridine nucleotide-cytochrome c reductase in liver.  J Biol Chem 1950, 183:593-605
  2. Williams CH Jr, Kamin H.  Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver.  J Biol Chem 1962, 237:587-95.  Phillips AH, Langdon RG.  Hepatic triphosphopyridine nucleotide-cytochrome c reductase: Isolation, characterization, and kinetic studies.  J Biol Chem 1962, 237:2652-60
  3. Lu AY, Coon MJ.  Role of hemoprotein P-450 in fatty acid omega-hydroxylation in a soluble enzyme system from liver microsomes.  J Biol Chem 1968 Mar 25 243:6 1331-2.  Lu AY, Junk KW, Coon MJ.  Resolution of the cytochrome P-450-containing omega-hydroxylation system of liver microsomes into three components.  J Biol Chem 1969 Jul 10 244:13 3714-21
  4. Enoch HG, Strittmatter P.  Cytochrome b5 reduction by NADPH-cytochrome P-450 reductase.  J Biol Chem 1979 Sep 25 254:18 8976-81. Ilan Z, Ilan R, Cinti DL.  Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system.  J Biol Chem 1981 Oct 10 256:19 10066-72
  5. Schacter BA, Nelson EB, Marver HS, Masters BS.  Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system.  J Biol Chem 1972 Jun 10 247:11 3601-7
  6. Ono T, Bloch K.  Solubilization and partial characterization of rat liver squalene epoxidase.  J Biol Chem 1975 Feb 25 250:4 1571-9.  Nishino H, Ishibashi T.  Evidence for requirement of NADPH-cytochrome P450 oxidoreductase in the microsomal NADPH-sterol Delta7-reductase system.  Arch Biochem Biophys 2000 Feb 15;374(2):293-8 [abstract]
  7. Taira Y, Greenspan P, Kapke GF, Redick JA, Baron J.  Effects of phenobarbital, pregnenolone-16alpha-carbonitrile, and 3-methylcholanthrene pretreatments on the distribution of NADPH-cytochrome c (P-450) reductase within the liver lobule.  Mol Pharmacol 1980 Sep 18:2 304-12.  Gonzalez FJ, Kasper CB.  Phenobarbital induction of NADPH-cytochrome c (P-450) oxidoreductase messenger ribonucleic acid.  Biochemistry 1980 Apr 29 19:9 1790-6.  Hardwick JP, Gonzalez FJ, Kasper CB.  Transcriptional regulation of rat liver epoxide hydratase, NADPH-Cytochrome P-450 oxidoreductase, and cytochrome P-450b genes by phenobarbital.  J Biol Chem 1983 Jul 10 258:13 8081-5 [abstract].  Simmons DL, McQuiddy P, Kasper CB.  Induction of the hepatic mixed-function oxidase system by synthetic glucocorticoids. Transcriptional and post-transcriptional regulation.  J Biol Chem 1987 Jan 5 262:1 326-32 [abstract]
  8. Waxman DJ, Morrissey JJ, Leblanc GA.  Hypophysectomy differentially alters P-450 protein levels and enzyme activities in rat liver: pituitary control of hepatic NADPH cytochrome P-450 reductase.  Mol Pharmacol 1989 Apr;35(4):519-25 [abstract].  Ram PA, Waxman DJ.  Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues.  Regulation by multiple mechanisms.  J Biol Chem 1992 Feb 15;267(5):3294-301 [abstract].   Li HC, Liu D, Waxman DJ.  Transcriptional induction of hepatic NADPH: cytochrome P450 oxidoreductase by thyroid hormone.  Mol Pharmacol 2001 May;59(5):987-995 [abstract]
  9. Dee A, Carlson G, Smith C, Masters BS, Waterman MR.  Regulation of synthesis and activity of bovine adrenocortical NADPH-cytochrome P-450 reductase by ACTH.  Biochem Biophys Res Commun 1985 Apr 30;128(2):650-6 [abstract]
  10. Simmons DL, Lalley PA, Kasper CB.  Chromosomal assignments of genes coding for components of the mixed-function oxidase system in mice. Genetic localization of the cytochrome P-450PCN and P-450PB gene families and the nadph-cytochrome P-450 oxidoreductase and epoxide hydratase genes.  J Biol Chem 1985 Jan 10;260(1):515-21 [abstract].  Shephard EA, Phillips IR, Santisteban I, West LF, Palmer CN, Ashworth A, Povey S.  Isolation of a human cytochrome P-450 reductase cDNA clone and localization of the corresponding gene to chromosome 7q11.2.  Ann Hum Genet 1989 Oct;53 ( Pt 4):291-301 [abstract]
  11. Porter TD, Beck TW, Kasper CB.  NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein.  Biochemistry 1990 Oct 23;29(42):9814-8 [abstract]
  12. O'Leary KA, Beck TW, Kasper CB.  NADPH cytochrome P-450 oxidoreductase gene: identification and characterization of the promoter region.  Arch Biochem Biophys 1994 May 1;310(2):452-9 [abstract].  O'Leary KA, McQuiddy P, Kasper CB.  Transcriptional regulation of the TATA-less NADPH cytochrome P-450 oxidoreductase gene.  Arch Biochem Biophys 1996 Jun 15;330(2):271-80 [abstract].  O'Leary KA, Kasper CB.  Molecular basis for cell-specific regulation of the NADPH-cytochrome P450 oxidoreductase gene.  Arch Biochem Biophys 2000 Jul 1;379(1):97-108 [abstract]
  13. O'Leary KA, Li HC, Ram PA, McQuiddy P, Waxman DJ, Kasper CB.  Thyroid regulation of NADPH:cytochrome P450 oxidoreductase: identification of a thyroid-responsive element in the 5'-flank of the oxidoreductase gene.  Mol Pharmacol 1997 Jul;52(1):46-53 [abstract]

 

 

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Comments to Todd D. Porter, Pharmaceutical Sciences, University of Kentucky College of Pharmacy, Lexington, KY 40536-0082.  Phone 859 257-1137; FAX 859 257-7564
Last Modified: November 12, 2001
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