P450c17 is the single enzyme mediating both 17a-hydroxylase (steroid 17ai-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. We sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, Xhacl7-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17. Comparison of the amino acid sequence of P450c17 with two other human steroidogenic cytochromes P450 show much greater homology with P450c21 (28.9%), another microsomal enzyme, than with P450scc (12.3%), a mitochondrial enzyme.Steroid 17a-hydroxylase (steroid 17a-monooxygenase, EC 1.14.99.9) converts pregnenolone to 17-hydroxypregnenolone and converts progesterone to 17-hydroxyprogesterone. These 17-hydroxylated steroids may then be converted by 17,20-lyase to dehydroepiandrosterone and androstenedione, respectively. These latter two steroids are precursors of testosterone and estrogen synthesis while 17-hydroxyprogesterone is a key precursor of cortisol synthesis. Although steroid 17a-hydroxylase and 17,20 lyase activities can be readily distinguished by examination of circulating venous steroidal products (1, 2), studies in both the guinea pig (3) and pig (4) show that both activities reside in a single protein, P450c17. Thus, the P450c17 enzyme is a key branch point in human steroid hormone synthesis, as 17a-hydroxylase activity distinguishes between synthesis of mineralocorticoids (aldosterone) and glucocorticoids (cortisol) and as 17,20 lyase activity distinguishes between synthesis of glucocorticoids and sex steroids. P450c17 is encoded by a gene or genes now termed P45OXVII (5). P450c17 mRNA accumulation is regulated hormonally (6, 7) and developmentally (8). Like P450c21 (steroid 21-hydroxylase), P450c17 is bound to the endoplasmic reticulum and accepts electrons f...

P450c17 is a single cytochrome P450 enzyme mediating both 17 alpha-hydroxylase and 17,20 lyase activities in the biosynthesis of steroid hormones. We have cloned and sequenced the human P450XVIIA1 gene lying on chromosome 10, which encodes P450c17. The gene spans 6569 bp and is divided into eight exons by seven introns. This intron/exon structure closely resembles that of the P450XXI genes encoding P450c21 (steroid 21-hydroxylase), which contain 10 exons, except that the introns dividing exons 1 and 2 and exons 4 and 5 in the P450XXI gene are absent in the P450XVII gene. Furthermore, computer modeling studies indicate the conformations of P450c17 and P450c21 are very similar. The structures of the P450XXVII and P450XXI genes are very different from other classes of P450 genes. Although the production of P450c17 is under different hormonal, ontogenic, and tissue-specific controls in various types of steroidogenic cells, the adrenal and testis transcribe the P450XVIIA1 gene into P450c17 mRNAs having the same cap sites. S1 nuclease protection experiments locate the principal cap sites as a G residue lying 22 bases 3' to an atypical TTTAAA promoter, and 82 bases 3' to a typical CAAT box. The 5'-flanking DNA contains sequences similar to consensus sequences regulated by cAMP and glucocorticoids.

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20 lyase activities. We identified several human P450c17 cDNA clones in a human adrenal cDNA library we constructed in lambda gt10. A short clone containing the 3'-terminal 650 bases of the full-length sequence was used to examine Southern blots of DNA from normal persons and from a panel of mouse/human somatic cell hybrid lines. The pattern of hybridization of this cDNA to normal human DNA cut with 8 restriction endonucleases suggests the human genome has two (or more) P450c17 genes. The pattern of hybridization to the somatic cell hybrid cell lines, each containing a limited, known number of human chromosomes, indicates the human adrenal P450c17 gene lies on chromosome 10. The chromosomal locations of the other P450c17 genes could not be determined.

Cytochrome P450c17 is the single microsomal enzyme catalyzing steroid 17 alpha-hydroxylase and 17-20-lyase activities. It is expressed and regulated by tropic hormones in the human adrenal and gonads, but is not expressed in the placenta. To study the transcriptional regulation of the human P450c17 gene, we constructed 11 plasmids containing serial deletions of its 5' nontranslated region driving expression of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into mouse adrenal Y1 and testis MA-10 cells and incubated with forskolin, 8-bromo-cAMP, or 12-O-tetradecanoyl-phorbol-13 acetate (TPA) for 12 h. Interpretation of results from standard constructions was difficult, apparently because some transcription was incorrectly initiated by DNA sequences in the vector. Therefore, we built a modified CAT reporter vector that eliminated detectable read-through transcription. In Y1 cells, the basal activity of constructs containing from -82 to -184 basepairs (bp) of 5' flanking DNA was between 80-150% of the promoterless control. Constructs containing at least -235 bp of this DNA expressed CAT at 540% of the control value, but addition of sequences to -774 had no further effect. Forskolin increased the expression of CAT activity to 300% above basal with constructions containing DNA from -184 to -774 bp. Constructs containing between -184 and -310 bp expressed CAT at 50% of the forskolin-induced levels in cells treated with TPA. Both basal and cAMP-induced expression were much lower in MA-10 cells than in Y1 cells and increased with increasing promoter length to -774.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenodoxin reductase is a mitochondrial flavoprotein that receives electrons from NADPH, thus initiating the electron-transport chain serving mitochondrial cytochromes P450. We have cloned and sequenced two human adrenodoxin reductase cDNAs that differ by the presence of six additional codons in the middle of one clone. The sequence in this region indicates that these six extra codons arise by alternative splicing of the pre-mRNA. Southern blot hybridization patterns of human genomic DNA cut with four restriction enzymes indicate that the human genome has only one gene for adrenodoxin reductase. Analysis of a panel of mousehuman somatic cell hybrids localized this gene to chromosome 17cen--q25. The alternatively spliced mRNA containing the six extra codons represents 10-20% of all adrenodoxin reductase mRNA. The expression of the adrenodoxin reductase gene may be stimulated by pituitary tropic hormones acting through cAMP, but its response is quantitatively much less than the responses of P450scc and adrenodoxin.The first and rate-limiting step in the synthesis of all steroid hormones is the conversion ofcholesterol to pregnenolone by a mitochondrial cytochrome termed P450scc. This cytochrome binds cholesterol and mediates three separate reactions on a single active site: 20-hydroxylation, 22-hydroxylation, and scission ofthe cholesterol side chain. Each ofthese reactions requires a pair of electrons. The electrons are transferred from NADPH to the flavoprotein adrenodoxin reductase and thence to the iron-sulfur protein adrenodoxin, which then donates them to the P450scc (1). This same electron-transport system donates electrons to another steroidogenic enzyme, P450c11 (2), to renal vitamin D lahydroxylase (3), and to hepatic 26-hydroxylase (4). The microsomal steroidogenic enzymes P450c17 (17a-hydroxylase/17,20-lyase) (5, 6), P450c21 (21-hydroxylase) (7,8), and P450aro (aromatase) (9) employ a different flavoprotein to transfer electrons from NADPH.

The amino acid sequences of two steroidogenic enzymes, P450c17 (steroid 17 alpha-hydroxylase/17,20 lyse) and P450c21 (steroid 21-hydroxylase), are only 28.9% identical. However, these proteins share a region of 21 amino acids bearing 17 identical residues, which we previously suggested may represent the steroid binding site. We assembled a sequence database of known steroid-binding proteins and searched this with the sequence of this 21 amino acid region. The steroidogenic enzymes, P450c17, P450c21, P450scc (the cholesterol side-chain cleavage enzyme), and P450c11 (steroid 11 beta/18-hydroxylase) share a subregion of 17 amino acids having at least 15 identical residues. Related sequences were identified in a computerized search of the available sequences of steroid hormone receptors and binding proteins. These sequences were invariably found within larger domains previously associated with steroid binding. From these we propose a more general consensus sequence of LPLLL +/- 000KDRE0LKRL +/- PV, where +/- refers to any charged amino acid, and 0 refers to an uncharged amino acid. This consensus sequence predicts 147 or 187 total amino acids in 11 human proteins examined (78.6%). An equivalent degree of sequence identity, 178 of 221 amino acids (80.5%) was found among 13 animal homologs of these human proteins. The ability of this consensus sequence to predict 325 of 408 amino acids (79.7%) strongly suggests this sequence is necessary, if not sufficient, for a steroid binding site in many proteins. Lecithin-cholesterol acetyl transferase, cholesterol ester transfer protein, and steroid sulfatase did not have sequences similar to our consensus sequence.

Adrenodoxin is an iron-sulfur protein found in the mitochondria of steroidogenic tissues. It participates in steroidogenesis as an electron transport intermediate for mitochondrial cytochromes P450, including P450scc, the cholesterol side-chain cleavage enzyme. Using a human adrenodoxin cDNA probe recently cloned in our laboratory, we examined the distribution and hormonal regulation of adrenodoxin mRNA in a variety of steroidogenic tissues. Adrenodoxin mRNA was found in all steroidogenic tissues examined. In human fetal testes, adrenodoxin mRNA was more abundant in early gestation, diminishing toward midterm in a pattern closely similar to that we reported previously for P450scc. Unlike P450scc, however, significant amounts of adrenodoxin mRNA were detected in human fetal ovaries, with no discernible gestation-dependent change. The abundance of adrenodoxin mRNA was increased in cultured human granulosa cells by treatment with hCG, FSH, cAMP, and cholera toxin. In human fetal adrenal cells, ACTH and cAMP stimulated accumulation of adrenodoxin mRNA, while in cultured human fetal testicular cells and cultured fetal rhesus monkey ovarian cells, both hCG and cAMP stimulated accumulation of adrenodoxin mRNA. In all of these systems, the accumulation of adrenodoxin mRNA closely paralleled the response of P450scc. These data suggest that the genes for these functionally related but structurally unrelated proteins are regulated in a coordinate manner.

No abstract