Introduction: transcription and regulation of activities of cytochromes P450 metabolizing endogenous substrates.
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Animals↗
Biomedical subjects
M R Waterman
Publications and source records attributed to M R Waterman.
P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.
We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), representing "cytochrome P450', be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.
Alleles↗
Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain.
Using the reverse transcription polymerase chain reaction, mRNAs encoding steroidogenic P450s as well as NADPH-cytochrome P450 reductase (P450 reductase), adrenodoxin and the transcription factor steroidogenic factor 1 (SF-1) were all detected in rodent brain, but their distribution between brain regions varied. Adrenodoxin and P450 reductase were detected in all regions, suggesting the presence of both mitochondrial and microsomal P450s throughout the brain. Messenger RNAs encoding P450scc (CYP11A1) and P45017 alpha (CYP17) were also detected in all brain regions, this being the first report of CYP17 in the brain. P450c21 (CYP21) was detected only in the brain stem. P45011 beta (CYP11B1) and P450aldo (CYP11B2) are expressed in rat brain, but not in mouse brain; CYP11B1 primarily in the cerebrum, whereas CYP11B2 was detected in all brain regions. In both species, highest levels of aromatase P450 (CYP19) mRNA were detected in the cerebrum. SF-1 expression was restricted to the cerebrum minus cortex. Thus, although SF-1 is required for high level expression of the steroidogenic enzymes in adrenals and gonads, other factors may influence the expression of these genes in the brain. If the mRNAs detected by RT-PCR are indeed translated into functional enzymes, these studies suggest that different brain regions have different capacities for local steroid hormone production and metabolism. This raises the technical challenge of locating the specific sites of synthesis as well as the function of such locally produced ligands.
Adrenodoxin↗
Protein kinase A-dependent transactivation by the E2A-Pbx1 fusion protein.
The chimeric gene E2A-PBX1 is formed by the t(1;19) chromosomal translocation exclusively associated with pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL). The resultant fusion protein from this chimeric gene contains the DNA-binding homeodomain of Pbx1. The first and only functional Pbx1 binding site has been localized in bovine CYP17 to a sequence (CRS1) that participates in cAMP-dependent transcription of this gene encoding the steroid hydroxylase, 17 alpha-hydroxylase cytochrome P450. Because Pbx1 is not expressed in pre-B cells, it may be possible that the E2a-Pbx1 fusion protein expressed in pre-B cells having this translocation will activate, in response to cAMP, transcription of genes not normally expressed in these cells leading to arrest of differentiation at the pre-B cell stage. We have now shown that reporter genes comprising CRS1 are activated transcriptionally by protein kinase A (PKA) in the pre-B cell line 697, which endogenously expresses the fusion protein, and that overexpression of E2A-Pbx1 in additional cell lines enhances transcription of reporter genes in a PKA-dependent fashion. Thus, it seems plausible that arrest in the pre-B stage leading to pre-B ALL includes cAMP-dependent activation of E2A-Pbx1.
Animals↗
Two Sp1-binding sites mediate cAMP-induced transcription of the bovine CYP11A gene through the protein kinase A signaling pathway.
Two sequence elements located at -111 to -100 base pairs and -70 to -50 base pairs in the 5'-flanking region of the bovine CYP11A gene and in closely related positions in CYP11A of other species contain G-rich regions that are similar to the consensus Sp1-binding site. These sequences bind the purified transcription factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that interact with an antibody specific for Sp1. Both of these CYP11A sequences support basal and cAMP-dependent transcription of reporter gene plasmids transfected into Y1 cells, and mutations within the G-rich -111/-100-base pair sequence that reduce or eliminate the binding of Sp1-related Y1 nuclear proteins also markedly reduce cAMP-induced transcription. cAMP-dependent transcription supported by both CYP11A sequence elements is mediated by protein kinase A at levels comparable to that promoted by different cAMP-response sequences and transcription factors in other genes involved in steroidogenesis. These results indicate that ACTH-dependent regulation of cholesterol side chain cleavage cytochrome P450 levels in the adrenal cortex which is mediated through cAMP involves the ubiquitous transcription factor Sp1.
Adrenal Cortex↗
Active-site topology of bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) and evidence for interaction of tyrosine 94 with the side chain of cholesterol.
Combining site-directed mutagenesis with analysis of the active-site topology of bovine cholesterol side-chain cleavage cytochrome P450scc (P450scc), we have investigated the roles of tyrosine residues 93 and 94 on substrate binding. Four single mutants (Y93A, Y93S, Y94A, and Y94S) and one double mutant (Y93S/Y94S) were examined. The largest increase in Ks was observed for binding of cholesterol and 25-hydroxycholesterol to the Y94S mutant (approximately 5.5-fold), with a smaller increase (< 2.5-fold) for binding of 22-hydroxycholesterol. Mutation of Y94 thus appears to influence the interaction with cholesterol, 25-hydroxycholesterol, and possibly 22-hydroxycholesterol. Y93 is not involved in binding of 22- and 25-hydroxycholesterol but may interact with cholesterol. The active-site topologies of P450scc and its mutants were probed by reaction with three arylhydrazines. The N-arylprotoporphyrin IX regioisomer patterns obtained with phenyl- and 2-naphthylhydrazine indicate that the active site is primarily open above pyrrole ring A and suggest that a region some distance above pyrrole ring D is also open. The single mutations Y93S, Y93A, Y94A, and Y94S do not detectably alter the regioisomer patterns obtained with the phenyl- and 2-naphthyl probes, but a small, reproducible change is observed with the 2-naphthyl probe for the Y93S/Y94S double mutant. The conformational alteration implied by this change could not be detected by titration with 22- and 25-hydroxycholesterol but is detectable by titration with cholesterol. The results indicate that cholesterol binds over pyrrole ring D of the heme in bovine P450scc, strongly suggest that Y94 interacts with the side chain of cholesterol, and provide evidence that the side chains of 22- and 25-hydroxycholesterol bind to a different region of the active site than the side chain of cholesterol.
Amino Acid Sequence↗
A rising StAR: an essential role in cholesterol transport.
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Biological Transport↗
A full-length cDNA encoding mouse adrenodoxin.
A full-length cDNA encoding mouse adrenodoxin was isolated from a Y1 adrenocortical tumor cell lambda ZAP cDNA library. The 883 bp cDNA contains a 567 bp open reading frame encoding a protein containing 188 amino acids. Mouse adrenodoxin shows high amino acid sequence identity with other mammalian adrenodoxins and the four cysteines involved in the formation of the iron-sulfur cluster are present.
Adrenal Gland Neoplasms↗
The role of cytochrome b5 in the biosynthesis of androgens by human P450c17.
Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17. It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to androstenedione by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b5. Other activities catalyzed by P450c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5. The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged. These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis. In particular, they indicate that in human testes which contains a high b5/P450 ratio, 17 alpha-hydroxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the delta 4 and the delta 5 steroidogenic pathways can lead to testosterone production.
17-alpha-Hydroxypregnenolone↗
Genetically engineered bacterial cells and applications.
Heterologous expression systems have proven to be very important in P450 research, permitting investigation of many P450s identified only by recombinant (cloning) technologies. They also make it possible to study human P450s since tissue sources for naturally occurring enzymes are difficult to obtain. A variety of different heterologous systems have been applied to the study of P450s and each one has its own unique advantages. For the study of biophysical properties and structure/function relationships, E. coli has proven to be a particularly good system. Both microsomal and mitochondrial P450s can be expressed to high levels in E. coli and subsequently purified to homogeneity for detailed analysis. Techniques of both site directed mutagenesis and random chimeragenesis are very facile in bacteria providing excellent opportunity to analyze specific aspects of structure/function relationships of P450s. Furthermore microsomal P450s are active in intact E. coli, activities being supported by flavodoxin and flavodoxin reductase, providing the opportunity to develop bioreactors expressing designer P450s. The salient features of P450 expression in bacteria are summarized herein.
Amino Acid Sequence↗
Developmentally regulated expression of adrenal 17 alpha-hydroxylase cytochrome P450 in the mouse embryo.
Corticosterone is the major circulating glucocorticoid in adult mice and rats, and this is explained, in part, by the absence of 17 alpha-hydroxylase cytochrome P450 (P450c17) in adrenal glands of these rodents. During embryonic development, however, we discovered transient expression of P450c17 in a subset of adrenocortical cells in the fetal mouse adrenal. This differs from cholesterol side-chain cleavage cytochrome P450 and adrenodoxin, which are expressed continuously in most fetal adrenocortical cells from onset of expression at embryonic days 11-12 (E11-12) until term. Adrenal P450c17 transcripts are detectable in situ at E12.5 and increase in abundance from E12.5 to E14.5. Transcripts are then lost between E16.5 and term (E18.5) and are undetectable in situ in adrenal glands of adult mice. These results are consistent with the presence of pregnenolone 17 alpha-hydroxylase activity in adrenal homogenates of fetal but not adult mice. By using polymerase chain reaction, we determined that murine fetal (E14.5-15.5) adrenal glands contain messenger RNAs (mRNAs) encoding all of the steroid hydroxylases required to produce cortisol and corticosterone but little aldosterone synthase mRNA. Adrenal glands from adult mice contain mRNAs encoding steroid hydroxylases required to produce corticosterone and aldosterone but not cortisol (little P450c17 mRNA). The spatial and temporal expression patterns of P450c17 and aldosterone synthase mRNA, which differ from those of cholesterol side-chain cleavage cytochrome P450 and adrenodoxin, suggest that multiple factors must be required to program cell type- and species-specific expression of these steroid hydroxylases during embryonic development.
Adrenal Glands↗
Cholesterol side-chain cleavage cytochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1.
In situ hybridization studies reveal novel sites of expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) during murine embryonic development. In addition to fetal adrenals and testes, P450scc transcripts localize in situ to the primitive gut and to a subset of unidentified cells in the dermal mesenchyme of embryonic skin. In the gut, transcripts are most abundant in luminal epithelia of the hindgut, which will form the colon. P450scc transcript abundance at these novel sites is a fraction of that in fetal adrenals or testes, suggesting a local rather than an endocrine function. Immunocytochemical analyses localize P450scc protein to the fetal hindgut, indicating that the transcripts are translated in vivo. RNA isolated from microdissected embryonic hindgut and skin was reverse transcribed and amplified by polymerase chain reaction. DNA sequence analyses of polymerase chain reaction products confirmed that specific hybridization in situ represents authentic P450scc gene (Cyp11A) transcripts and that 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4-isomerase transcripts are also present, demonstrating the potential of these fetal tissues to produce pregnenolone and progesterone. P450scc transcripts are also detectable by in situ hybridization in primitive gut and skin of Fushi tarazu factor 1 null mice, which lack the nuclear receptor steroidogenic factor 1, proving that steroidogenic factor 1 is not required for steroid hydroxylase gene expression at these sites. The capacity for C21 steroid biosynthesis in primitive gut and skin during organogenesis raises the question whether local production of steroid hormones may be required for normal cellular growth and differentiation of these tissues during embryogenesis.
Animals↗
Biochemical diversity of cAMP-dependent transcription of steroid hydroxylase genes in the adrenal cortex.
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Adrenal Cortex↗
Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities.
Two soluble flavoproteins, purified from Escherichia coli cytosol and identified as flavodoxin and NADPH-flavodoxin (ferredoxin) reductase (flavodoxin reductase), have been found in combination to support the 17 alpha-hydroxylase activities of heterologously expressed bovine 17 alpha-hydroxylase cytochrome P450 (P450c17). Physical characteristics of the two flavoproteins including absorbance spectra, molecular weights, and amino-terminal sequences are identical with those reported previously for E. coli flavodoxin and flavodoxin reductase. Flavodoxin reductase, possessing FAD as a cofactor, is able to reconstitute P450c17 activities only in the presence of flavodoxin, an FMN-containing protein, and NAD(P)H. Reducing equivalents are utilized more effectively from NADPH than NADH by flavodoxin reductase. E. coli flavodoxin binds P450c17 directly and with relatively high affinity (apparent Ks approximately 0.2 microM) at low ionic strength, as evidenced by a change in spin state of the P450c17 heme iron upon titration with flavodoxin. This apparent spin shift is attenuated at moderate ionic strengths (100-200 mM KCl). In addition, bovine P450c17 binds reversibly to flavodoxin Sepharose in an ionic strength-dependent manner. These data implicate charge pairing as being important for the interaction between flavodoxin and P450c17. We propose that the amino acid sequence similarity between E. coli flavodoxin-flavodoxin reductase and the putative FMN, FAD, and NAD(P)H binding regions of cytochrome P450 reductase provides the basis for the reconstitution of P450c17 activities by this bacterial system.
Aldehyde-Lyases↗
A cAMP-regulatory sequence (CRS1) of CYP17 is a cellular target for the homeodomain protein Pbx1.
Cytochrome P450c17 encoded by CYP17, whose expression is regulated by peptide hormones via cAMP, is required for cortisol and sex hormone biosynthesis thereby playing a key role in biological processes including sexual differentiation. Utilizing the cAMP-regulatory sequence CRS1 of the bovine CYP17 gene as an affinity ligand, four CRS1-binding proteins have been purified from nuclear extracts of mouse adrenocortical Y1 cells and shown to enhance the in vitro transcription of a reporter gene promoted by CRS1. Microsequencing of these four proteins established two of them to be the homeodomain proteins Pbx1a and Pbx1b, originally discovered by their involvement in the t(1;19) chromosomal translocation in pre-B-cell acute lymphoblastic leukemias. Overexpression of Pbx1 in Y1 cells enhances cAMP-dependent transcription of the CRS1-dependent reporter gene. These results identify the CRS1 of bovine CYP17 as a cellular target for Pbx1 and suggest that one role of this homeodomain protein is in the regulation of steroidogenesis and subsequently sexual development.
Amino Acid Sequence↗
Heterologous expression of mammalian P450 enzymes.
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Amino Acid Sequence↗
Baculovirus expression of bovine cytochrome P450c17 in Sf9 cells and comparison with expression in yeast, mammalian cells, and E. coli.
Expression of bovine 17 alpha-hydroxylase cytochrome P450 (P450c17) in insect Sf9 cells using baculovirus is the fourth heterologous expression system developed for studying this enzyme. The enzyme in Sf9 cell membranes has all the expected activities and closely resembles that from the other systems: COS1 cells, yeast, and Escherichia coli. One curious feature of baculovirus expression of this hemoprotein is that the immunodetectable P450c17 is present in an insoluble pellet when produced in the absence of added hemin and in a 10,000 g supernatant when expressed in the presence of added hemin. When comparing the level of expression of bovine P450c17 in these different expression systems, E. coli produces the largest quantity per liter culture, while baculovirus produces the most molecules of P450c17 per cell.
Animals↗
No mutation in cytochrome P450 side chain cleavage in a patient with congenital lipoid adrenal hyperplasia.
Molecular basis of lipoid adrenal hyperplasia (lipoid CAH) in a Japanese patient was investigated. A 46XY Japanese female patient was clinically diagnosed as having lipoid CAH based on her clinical history of adrenal crisis at birth and the low basal concentrations of cortisol, aldosterone, adrenal androgens and testosterone in serum. In vitro studies of testicular mitochondrial enzymes confirmed a specific impairment of cholesterol side chain cleavage (SCC) activity. However, in spite of the virtual reduction of SCC activity, the amounts of immunodetectable P450scc, adrenodoxin reductase, and adrenodoxin in testicular mitochondria were almost same as those of normal testis. Furthermore, the size of each protein was similar to that of normal testis. Enzymatic amplification of the complementary DNA encoding P450scc from the patient's testis RNA and its nucleotide analysis by direct sequencing revealed no mutation. These results indicate that defective P450scc is not the lesion in this patient, confirming a previous report showing no P450scc mutations in patients with lipoid CAH. The exact lesion causing lipoid CAH in this patient is currently unknown.
Adolescent↗