Characterization and tissue distribution of a novel human cytochrome P450—CYP2U1

doi:10.1016/j.bbrc.2004.01.110

Biochemical and Biophysical Research Communications

Volume 315, Issue 3, 12 March 2004, Pages 679-685

https://doi.org/10.1016/j.bbrc.2004.01.110 Get rights and content

Abstract

A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.

Section snippets

Materials and methods

Bioinformatics. The BLASTN and TBLASTN search algorithms [11] were used to search the htgs database [12] for sequences related to previously described CYP2 sequences. The amino acid sequence of CYP2U1 was compared to the amino acid sequences of other human P450s using multiple sequence alignment. An unrooted phylogenetic tree was calculated using ClustalW 1.8 [13] and visualized using the program “unrooted” (http://pbil.univ-lyon1.fr/software/unrooted.html).

cDNA preparation, amplification, and

Bioinformatics

When searching the htgs database two sequences (GenBank Accession Nos. AC025090 and AC000016) were identified that contained parts of a novel human CYP2 sequence that later turned out to be equivalent to CYP2U1 (http://drnelson.utmem.edu/CytochromeP450.html). The open reading frame obtained from these two sequences was confirmed by amplification of the CYP2U1 cDNA from human liver followed by sequencing. The open reading frame is 1635 nucleotides long and encodes a 544 amino acid long

Conclusions

The high conservation across species as well as the selective tissue distribution suggests that CYP2U1 has important endogenous functions in thymus and brain. Other brain-specific P450 are involved in the metabolism of steroid hormones, fatty acids or cholesterol. The murine cyp2j9, which is expressed in cerebellar Purkinje cells, is metabolizing arachidonic acid [7], while CYP46 is involved in the metabolism of cholesterol [6]. The endogenous function of CYP2U1 is still unknown but it is

Acknowledgements

This work was supported by a grant from The Swedish Research Council. The authors thank Johan Ställberg for his valuable contribution in Western blot analysis.

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Hereditary spastic paraplegia 56 (SPG56) is an extremely rare autosomal recessive disorder caused by mutations in the CYP2U1 gene, involved in fatty acid metabolism. SPG56 causes progressive spasticity in upper and lower limbs, though due to the rarity of this subtype of spastic paraplegia, the molecular causes remain unclear and no treatment or cure exists. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from two unrelated patients with SPG56 and two heterozygous family members. These lines can be used to investigate the mechanisms driving progressive spasticity and evaluate the potential for gene replacement therapies.
Bta-miR-130a/b regulates preadipocyte differentiation by targeting PPARG and CYP2U1 in beef cattle
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Deposition of intramuscular fat (IMF) is one of the most important traits for the evaluation of beef carcass quality grade. MicroRNA (miRNA) is an endogenous non-coding RNA that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. Previously, we identified that bta-miR-130a regulates milk fat biosynthesis by targeting PPARG mRNA. However, the role of miR-130 in the regulation of bovine adipocyte differentiation remains unknown. In this study, we found that overexpression of bta-miR-130a/b led to significantly decreased cellular triacylglycerol (TAG) levels during adipogenesis process as well as reduced lipid droplet formation. In contrast, the inhibition of bta-miR-130a/b resulted in larger lipid droplets and TAG accumulation. In addition, overexpression of bta-miR-130a/b inhibited the expression of adipocyte differentiation-related genes, including PPARG, C/EBPα, C/EBPβ, FABP4, LPIN1, and LPL. Western blot analysis verified qPCR results on the expression of PPARG and CYP2U1. A luciferase reporter assay further verified bta-miR-130a/b significantly affects PPARG and CYP2U1 expression by directly binding to their 3′-untranslated regions (UTR). We conducted in vitro rescue assay to confirm that bta-miR-130a/b affect bovine adipocyte differentiation by targeting PPARG and CYP2U1. This study shows that bta-miR-130a and bta-miR-130b play similar roles in the regulation of adipocyte differentiation in beef muscles by targeting the 3‘UTR of PPARG and CYP2U1. Our result provides a reference for illustrating how noncoding RNAs affects beef quality traits in cattle.
Membrane-bound human orphan cytochrome P450 2U1: Sequence singularities, construction of a full 3D model, and substrate docking
2017, Biochimie
Citation Excerpt :
Among them, CYP2U1 exhibits several surprising, distinctive characteristics [4,5]. With 544 amino acids, it is by far the longest isoform of family 2 with more than 40 extra residues as compared to the other CYP2s [6,7]. Also, with only five exons, the CYP2U1 gene, as the CYP2R1 gene, differs from the typical CYP2 splicing pattern which presents nine exons with the intron–exon boundaries preserved in location and phase [8].
Human cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms.
We compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model.
The protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A′ and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA′ and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities.
This model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation.
Spectral and 3D model studies of the interaction of orphan human cytochrome P450 2U1 with substrates and ligands
2017, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
Among them, CYP2U1 is highly conserved among the living kingdom [5–8]. Studies of its distribution have shown that it is preferentially expressed in the thymus and cerebellum, even though its presence was also detected in the kidneys, lungs, heart, white adipose tissue, platelets, and the blood-brain barrier [5,6,9–16]. CYP2U1 was found to be up-regulated in a variety of cancer tissues such as breast or colorectal cancer tissues [17,18].
Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an “orphan” protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine.
We have expressed CYP2U1 in E. coli and performed UV–vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS.
The UV–vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate Fe^III state. Imidazole (Im) derivatives, such as miconazole, acted as Fe^III ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to “reverse type I” difference UV–vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model.
Our study describes for the first time the mode of interaction of several Fe^III-heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP.
Expression in yeast, new substrates, and construction of a first 3D model of human orphan cytochrome P450 2U1: Interpretation of substrate hydroxylation regioselectivity from docking studies
2015, Biochimica et Biophysica Acta - General Subjects
Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an “orphan” enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin.
We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography–mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions.
We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site.
Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model.
These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.
Oxidation of endogenous N-arachidonoylserotonin by human cytochrome P450 2U1
2014, Journal of Biological Chemistry
Citation Excerpt :
The IC50 of 2-oxo-N-arachidonoylserotonin in the same system was 47 ± 5 μm. P450 2U1 was first identified in 2004 through searches of the human genome (17). The closest human relatives are P450s 2R1 (37% identity) and 2J2 (36% identity).
Cytochrome P450 (P450) 2U1 has been shown to be expressed, at the mRNA level, in human thymus, brain, and several other tissues. Recombinant P450 2U1 was purified and used as a reagent in a metabolomic search for substrates in bovine brain. In addition to fatty acid oxidation reactions, an oxidation of endogenous N-arachidonoylserotonin was characterized. Subsequent NMR and mass spectrometry and chemical synthesis showed that the main product was the result of C-2 oxidation of the indole ring, in contrast to other human P450s that generated different products. N-Arachidonoylserotonin, first synthesized chemically and described as an inhibitor of fatty acid amide hydrolase, had previously been found in porcine and mouse intestine; we demonstrated its presence in bovine and human brain samples. The product (2-oxo) was 4-fold less active than N-arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The rate of oxidation of N-arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The demonstration of the oxidation of N-arachidonoylserotonin by P450 2U1 suggests a possible role in human brain and possibly other sites.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Bioinformatics

Conclusions

Acknowledgements

References (20)

A novel cytochrome P450 expressed primarily in brain

J. Biol. Chem.

Cytochrome P450 CYP2J9, a new mouse arachidonic acid omega-1 hydroxylase predominantly expressed in brain

J. Biol. Chem.

Identification of a novel cytochrome P450, CYP4X1, with unique localization specific to the brain

Biochem. Biophys. Res. Commun.

Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme

J. Biol. Chem.

The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature

J. Biol. Chem.

Protein measurements with the Folin phenol reagent

J. Biol. Chem.

The 2.6-Å crystal structure of Pseudomonas putida cytochrome P-450

J. Biol. Chem.

Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution

Arch. Biochem. Biophys.

Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants

Pharmacogenetics

Cytochrome P450 in the brain; a review

Curr. Drug Metab.

Cited by (57)

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Spectral and 3D model studies of the interaction of orphan human cytochrome P450 2U1 with substrates and ligands

Expression in yeast, new substrates, and construction of a first 3D model of human orphan cytochrome P450 2U1: Interpretation of substrate hydroxylation regioselectivity from docking studies

Oxidation of endogenous N-arachidonoylserotonin by human cytochrome P450 2U1