Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0338671 (Steroids)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The conversion of androgens into estrogen involves three distinct generic reactions which are catalyzed by a single P450 enzyme (aromatase or P450(aromatase)). The first step in the process is the conversion of 19-methyl into a hydroxymethyl group which requires NADPH + O2, thus representing the well-known hydroxylation process. The next stage, converting the -CH2OH into -CHO, also requires NADPH + O2 and may be rationalized either through a second hydroxylation reaction producing a gem-diol, CH(OH)2 (which dehydrates to the aldehyde), or via another route. The final stage in the process again uses NADPH + O2, culminating in the release of C-19 as formate. Our extensive studies using precursors containing 2H, 3H, and 18O have shown that the carbonyl oxygen of the 19-aldehyde group is the one that was introduced in the first step as the hydroxyl group. The aldehydic oxygen along with another, from O2, used in the third step of the process, is incorporated into the released formate. It was found that at each stage of the process, oxygen atoms were introduced or transferred as "whole numbers." In light of these data, mechanisms in which H2O is used to promote the C-10-C-19 bond cleavage or those in which the conversion of the 19-oxoandrostenedione into estrogen is considered to occur via the sequence -CHO----(-)CH(OH)2----estrogen are eliminated. In addition, our mechanistic analysis makes it unlikely that 1 beta-, 2 beta-, or 10 beta-hydroxysteroids serve as intermediates in estrogen biosynthesis. We consider a free radical mechanism for the hydroxylation process.
Steroids 1990 Apr
PMID:Studies on estrogen biosynthesis using radioactive and stable isotopes. 218 83

To elucidate the mechanisms of abnormal steroid production in hyperfunctioning and non-hyperfunctioning adrenal tumors, we examined both the activities and amounts of steroidogenic cytochromes P450 in the tumor and non-tumor portions of these adrenals at the posttranslational (protein) level. Adrenals from 5 patients with primary aldosteronism, 5 with Cushing's syndrome, 1 with deoxycorticosterone (DOC)-producing adenoma, 10 with non-hyperfunctioning adrenal adenoma, and 5 subjects with normal control adrenals (obtained from patients with renal cell carcinoma) were used in our studies. Activities of P450scc, P45011 beta and P450aldo, and P450C21 and P-45017 alpha were assayed in a reconstituted enzyme system using 20 alpha-hydroxycholesterol, DOC, and progesterone, respectively, and the substrate and the extracted products were analyzed by HPLC. Enzyme amounts were determined by immunoblot analysis with anti-bovine P450scc, P45011 beta, and P450C21 IgG, and anti-porcine P45017 alpha IgG. Human P450aldo was only detected in the tumor portion of primary aldosteronism adrenals, with both activities and amounts of other P-450s similar to those in the non-tumor portion of primary aldosteronism and normal controls. In Cushing's syndrome, both activities and amounts of P45017 alpha and P450C21 were significantly increased in the tumor compared with those in the non-tumor portion of Cushing's syndrome and normal controls. In DOC-producing adenoma, both activities and amounts of P45017 alpha and P45011 beta in the tumor portion of the adenoma decreased compared with normal control, while those of other P450s were similar to normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Steroids 1995 Jan
PMID:Significance of steroidogenic enzymes in the pathogenesis of hyperfunctioning and non-hyperfunctioning adrenal tumor. 779 14

Using cultured bovine adrenal fasciculata cells (BAC), we investigated the effects of two hormones, corticotropin (ACTH) and angiotensin II (Ang-II) and two growth factors, insulin-like growth factors I (IGF-I) and transforming growth factor beta 1 (TGF beta 1), on the mRNA levels of nuclear proto-oncogenes of the Fos and Jun families and on the mRNA levels of genes expressed in BAC coding for ACTH and AT1 receptors, cytochrome P450scc and P450 17 alpha and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). ACTH and IGF-1 increased c-fos and jun-B mRNA levels early with later increases in the levels of mRNA for the ACTH receptor and the three steroidogenic enzymes, and enhanced steroidogenic responses to both ACTH and Ang-II. In contrast, Ang-II increased mRNA coding for the three proto-oncogenes (cfos, c-jun, and jun-B), decreased those for P450 17 alpha and 3 beta-HSD, and caused marked homologous and heterologous steroidogenic desensitization. TGF beta 1 increased only jun-B mRNA and markedly reduced BAC-differentiated functions and steroidogenic responsiveness to both ACTH and Ang-II. The long-term effects of ACTH on human adrenal fasciculata cells were comparable with those observed in BAC, whereas the long term effects of Ang-II and TGF beta 1 were different from those observed in BAC. Whether these species-specific differences are related to a different effect of these factors on proto-oncogene expression is not yet known.
Steroids 1996 Apr
PMID:Regulation of primary response and specific genes in adrenal cells by peptide hormones and growth factors. 873 96

We produced a murine monoclonal antibody (MAb) to human placental aromatase cytochrome P450. This MAb, designated MAb3-2C2, was selected on its ability to suppress aromatase activity. The specificity of this MAb was assessed by selective immunoprecipitation of 125I-labeled aromatase cytochrome P450 as well as by the identification of a 55-kDa protein, which was enriched and purified by immunoaffinity chromatography on a MAb-coupled Sepharose 4B column. The MAb was able to suppress both human placental and ovarian microsomal aromatase. Species differences of aromatase were recognized by MAb3-2C2 on the basis of differential immunosuppression of aromatase activity. The antibody had no effect on non-aromatase cytochrome P450s. MAb3-2C2 gave negative results with human placental aromatase P450 in the Western blot analysis. The data presented indicate that MAb3-2C2 is specific for aromatase cytochrome P450 and that its epitope is located in a fragile tertiary conformation of the enzyme, thus making it capable of sensitively affecting catalysis.
Steroids 1996 Mar
PMID:Preparation of an activity-inhibiting monoclonal antibody against human placental aromatase cytochrome P450. 885 29

Steroids bearing a heteroaromatic substituent at C-17 were designed as inhibitors of C17(20) lyase. The thiazoles, furans, and thiophenes appended to the steroid nucleus were positioned on the alpha-face and the beta-face of the steroid, and conjugated with a 16,17-olefin, to test their ability to coordinate the heme iron of the P450 enzyme complex. The position of the heterocycle with respect to the steroid skeleton was determined to be important for optimum affinity and, in general, compounds with the heterocycle attached to a trigonal center at C-17, had the best affinity for C17(20) lyase. Simple molecular models were used to compare the three types of heterocyclic-substituted steroids.
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PMID:Inhibition of steroid C17(20) lyase with C-17-heteroaryl steroids. 889 99

Much of our understanding of P450 reaction mechanisms derives from studies on P450cam, a bacterial camphor hydroxylase. P450cam has served as the model for understanding detailed structure/function relationships in mammalian P450 enzymes, which have not proved amenable to x-ray crystallographic techniques. To expand and improve the P450 model, we solved the structure of P450eryF, a cytochrome P450 involved in erythromycin biosynthesis. The overall structure of P450eryF is similar to that of P450cam, but differs in the exact positioning of several alpha-helices, which results in the enlargement of the substrate-binding pocket. P450eryF also differs from P450cam in having alanine in place of the highly conserved threonine residue in the active site. To assess the role of this alanine residue, two mutant forms of P450eryF and a substrate analog were examined. Our findings suggest that P450eryF has evolved an active site that utilizes the substrate to assist in catalysis. In addition, the enlarged substrate binding pocket of P450eryF enables P450eryF to bind certain steroid compounds and azole-based steroid hydroxylase inhibitors. Crystals have been obtained for P450eryF complexed with the antifungal drug ketoconazole, and the high-resolution structure has been determined.
Steroids 1997 Jan
PMID:Structure of cytochrome P450eryF: substrate, inhibitors, and model compounds bound in the active site. 902 24

Over 400 P450s have been identified to date in prokaryotes and eukaryotes, plants and animals, mitochondria and endoplasmic reticulum. These enzymes function in areas such as metabolism and steroidogenesis. The eukaryotic members of this gene superfamily of proteins have proved difficult to study because of the hydrophobic nature of their substrates, their various redox partners, and membrane association. To better understand the structure/function relationship of P450s-what determines substrate specificity and selectivity, what determines redox-partner binding, and which regions are involved in membrane binding-we have compared the three crystallized, soluble bacterial P450s (two class I and one class II) and a model of a steroidogenic, eukaryotic P450 (P450arom), to define which structural elements form a conserved structural fold for P450s, what determines specificity of substrate binding and redox-partner binding, and which regions are potentially involved in membrane association. We believe that there is a conserved structural fold for all P450s that can be used to model those P450s that prove intransigent to structural determination. However, although there appears to be a conserved structural core among P450s, there is sufficient sequence variability that no two P450s are structurally identical. NADPH-P450 reductase transfers electrons from NADPH to P450 during the P450 catalytic cycle. This enzyme has usually been thought of as a simple globular protein; however, sequence analysis has shown that NADPH-P450 reductase is related to two separate flavoprotein families, ferredoxin nucleotide reductase (FNR) and flavodoxin. Recent studies by Wolff and his colleagues have shown that the FAD-binding FNR domain and FMN-binding flavodoxin domain of human NADPH-P450 reductase can be independently expressed in Escherichia coli. The subdomains can be used to reconstitute, however poorly, the monooxygenase activity of the P450 system. We have been utilizing the reductase domain of P450BM-3 to study the mechanism of electron transfer from NADPH to P450 in this complex multidomain protein. We have overexpressed both the FNR subdomain and the flavodoxin subdomain in E. coli and fully reconstituted the cytochrome c reductase activity of this enzyme. Our studies have shown that electron transfer from NADPH through the reductase domain to the P450 requires shuttling of the FMN subdomain between the reductase subdomain and the P450. Studies of the factors that control the molecular recognition and interaction among these three proteins are complicated by the weakness of the association and changes in the strength of the interaction depending on the redox state of each of the components. How these structural and mechanistic studies of a soluble bacterial P450 can be extended to gain a better understanding of the control of membrane-bound eukaryotic P450-dependent redox systems is discussed.
Steroids 1997 Jan
PMID:P450BM-3; a tale of two domains--or is it three? 902 25

Mitochondrial monooxygenase systems are involved in the biosynthesis of glucocorticoids, mineralocorticoids, bile acids, and 1,25-dihydroxyvitamin D. The reactions are catalyzed by specific P450 enzymes that receive reducing equivalents via NADPH-ferredoxin oxidoreductase (adrenodoxin reductase) and ferredoxin (adrenodoxin). Although the three-dimensional structures of the individual components have not yet been solved, methods of expressing recombinant forms of these enzymes in Escherichia coli have allowed the use of site-directed mutagenesis to investigate the roles of specific amino acids in protein binding interactions, electron transfer, and catalysis. These studies have identified key charged residues in NADPH-ferredoxin oxidoreductase, ferredoxin, and P450scc, which are involved in electrostatic interactions critical for recognition, high-affinity binding, and electron transfer. The finding that the binding sites on ferredoxin for NADPH-ferredoxin oxidoreductase and P450 show significant overlap supports the proposed function for ferredoxin as a mobile electron shuttle between the reductase and P450 enzymes and is consistent with ferredoxin's role in serving multiple P450 isoforms.
Steroids 1997 Jan
PMID:Molecular recognition and electron transfer in mitochondrial steroid hydroxylase systems. 902 26

P450c17 is a single microsomal enzyme that catalyzes two distinct steroid biosynthetic activities: 17 alpha-hydroxylase and 17,20 lyase. Human beings have only one gene that encodes only one form of P450c17. Three clinical observations indicated that these were independently regulated activities. First, several cases of isolated 17,20 lyase deficiency were reported, in which 17 alpha-hydroxylase activity was spared. Second, most adrenal steroidogenesis in children stops after 17 alpha-hydroxylation, thus permitting the synthesis of cortisol, whereas most gonadal steroidogenesis proceeds to C19 sex steroids as a result of both activities. Third, the 17,20 lyase activity of the human adrenal is developmentally activated during adrenarche. To catalyze these two activities, P450c17 must receive reducing equivalents from electron donors (redox partners). Previous observations showed that the molar ratio of P450 oxidoreductase to P450c17 was 3-fold higher in the testis than in the adrenal, and that increasing the molar ratio of the redox partner to P450c17 would increase the ratio of 17,20 lyase activity to 17 alpha-hydroxylase. We have recently shown that P450c17 must be phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase to acquire 17,20 lyase activity. We have also recently found two cases of isolated 17,20 lyase deficiency that have mutations of residues in the proposed redox partner binding site. Together, these studies suggest a unified view of the regulation of 17,20 lyase activity. The ratio of 17,20 lyase to 17 alpha-hydroxylase activity of P450c17 is regulated by the availability of reducing equivalents flowing to the enzyme. This can be increased by increasing the molar concentration of electron-donating redox partners, such as P450 oxidoreductase or possibly cytochrome b5, as appears to be the case in the gonads. Alternatively, the affinity of P450c17 for redox partners may be selectively increased by Ser/Thr phosphorylation, or selectively decreased by certain mutations in the redox partner binding site, in either case altering an electrostatic interaction between P450c17 and the redox partner. This model is consistent with all present observations about the biochemistry, genetics, enzymology, and clinical phenomenology of P450c17.
Steroids 1997 Jan
PMID:The regulation of 17,20 lyase activity. 902 28

Using transgenic mice, we targeted SV40 T antigen and the bacterial neomycin resistance gene to steroidogenic tissues using a human P450 cholesterol side-chain cleavage promoter. Expression of SV40 T antigen resulted in adrenocortical tumors. Adrenocortical cell lines from one of these tumors (ST5R) was previously characterized. We have now obtained clonal lines from the second more differentiated tumor. After dispersion of the left adrenal tumor, ST5L parental cells were selected with G418 and subcloned. The resulting adrenocortical subcloned cell lines are more highly differentiated than those cell lines resulting from the right adrenal tumor (ST5R). ST5L cell lines secrete progesterone and corticosterone to varying degrees, whereas ST5R cells secrete only progesterone. One of the clonal cell lines, ST5Lc16, expresses both P450c11 beta and P450c11AS mRNAs, which normally are regionally distributed in different zones of the adrenal cortex. Thus, ST5Lc16 cells may be progenitor cells for both glomerulosa and fasciculata cells and may provide clues to the cellular and molecular events leading to the differentiation of the glomerulosa and the fasciculata-reticularis. Other ST5Lc cell lines are more representative of the fasciculata-reticularis, because they express P450c11 beta mRNA and secrete corticosterone, and they neither express P450c11AS mRNA nor do they secrete aldosterone. All cell lines also have 21-hydroxylase activity, but none express P450c21, indicating that some other, as yet unidentified, enzyme has this activity. In all cell lines, steroid secretion is regulable by cAMP stimulation but not by ACTH stimulation. All ST5L cell lines also express mouse renin-1 mRNA. In addition to their utility in studies of adrenal steroidogenesis, these cell lines may also be useful in studying the etiology of adrenocortical tumors.
Steroids 1997 Feb
PMID:Characterization of adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice. 905 83


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