Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytochrome P450c17 is the single enzyme having steroid 17 alpha-hydroxylase and 17,20 lyase activities. We sought to model the active site of this enzyme to identify residues contributing to its catalytic activities, and to test the roles of the identified amino acids by altering them via site-directed mutagenesis. Using the MIDAS-plus program, we modeled P450c17 and the structurally related steroid 21-hydroxylase, P450c21, on the crystallographically determined structure of bacterial P450cam. By positioning the progesterone substrate into each model, we identified five residues that appeared crucial for determining whether progesterone would undergo 17 alpha-hydroxylation (by P450c17) or 21-hydroxylation (by P450c21). Each identified residue in the P450c17 sequence was changed to the corresponding residue in the P450c21 sequence, yielding the four P450c17 mutants L102Y, G111D, G301l, and M369L + l371L. The mutants were transfected into COS-1 cells and their 17 alpha-hydroxylase, 17,20 lyase, and 21-hydroxylase activities were assayed by incubation with [14C]pregnenolone, [3H]17OH-pregnenolone, and [3H]17OH-progesterone and TLC. The L102Y and M369L + l371L mutants retained 50-80% of 17 alpha-hydroxylase and 70-100% of 17,20 lyase activity, while the G111D and G301l mutants lost both activities, but no mutants acquired detectable 21-hydroxylase activity (0.1% of wild type P450c21). Combination of the two mutants that retained partial activity (L102Y and M369L + l371L) yielded a single protein that retained 40% of 17 alpha-hydroxylase and 50% of 17,20 lyase activity, but none of the seven possible vectors expressing two, three, or all four of the mutations in a single enzyme yielded detectable 21-hydroxylase activity. The mutations D298V and D298S were predicted to ablate 17,20 lyase activity while retaining 17 alpha-hydroxylase activity, but were both inactive. These studies indicate that models based on the crystal structure of P450cam correctly predict many gross architectural features of steroidogenic enzymes and that many of the predicted residues are in or near the active site of P450c17. However, because enzymatic activity requires interactions between the enzyme and substrate at distances of less than 1 A, and modeling cannot predict atomic loci to greater than 1.5-2.0 A, it was not possible to design mutants that would confer 21-hydroxylase activity to P450c17. Currently available data cannot predict the structural and amino acid sequence requirements for a specific P450 activity.
Mol Endocrinol 1994 Mar
PMID:Modeling and mutagenesis of the active site of human P450c17. 801 56

Congenital adrenal hyperplasia (CAH) results from an enzymatic block at any stage in the synthesis of cortisol. All enzyme defects causing CAH are autosomal recessive traits. It is a relatively common disease, occurring in 1 in 5000 to 1 in 15,000 births in most populations. Since the isolation of the gene responsible for steroid 21-hydroxylase deficiency (involved in about 90% of the cases of CAH) in 1984, knowledge of the specific mutations that cause the different forms of CAH has grown rapidly. Defects in the encoding gene have been confirmed as the basis of endocrine disease in the case of all but one of the adrenal steroidogenic enzymes. Analysis of DNA obtained by chorionic villus sampling in early pregnancy permits prenatal diagnosis and treatment of 21-hydroxylase deficiency CAH. The correlation between the clinical expression of endocrine disease and the mutations of the primary structural gene is not absolute. Clinicians cannot accurately predict the course of the disease or make therapeutic decisions based on the genotype alone. We will review the various forms of clinical presentation of 21-hydroxylase CAH, its etiology, diagnosis, molecular genetics, and treatment.
J Steroid Biochem Mol Biol 1994 Jan
PMID:21-hydroxylase deficiency congenital adrenal hyperplasia. 813 1

Studies of adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y-1 cells. We sought to make new, alternative mouse steroidogenic cell lines by genetically targeted tumorigenesis. Transgenic mice were constructed expressing both the SV40 T-antigen and a bacterial neomycin-resistance gene under the control of the promoter for the human P450 cholesterol side-chain cleavage (P450scc) gene, which encodes the first and rate-limiting enzyme in steroidogenesis. Two female transgenic mice expressed T-antigen in various nonsteroidogenic tissues but generated tumors only in the adrenals, suggesting adrenal tumor formation was an early event. Ovarian tissues, which, unlike the adrenal, do not make steroids in fetal or early postnatal life, did not develop tumors. Cell lines derived from the adrenal tumors were resistant to the neomycin analog G418. Clonal sublines are stable, growing easily in monolayers with a doubling time of 24-60 h. The cell lines secrete progesterone and 11-deoxycorticosterone, indicating these cells express the P450scc system, 3 beta-hydroxysteroid dehydrogenase, and 21-hydroxylase activity. However the 21-hydroxylase activity was not mediated by P450c21, as the cells lacked P450c21 mRNA. The cells did not secrete any 11-hydroxylated steroids, although they contained P450c11 beta mRNA. Both the secretion of progesterone and the abundance of P450scc mRNA increase in response to 8-bromo-cAMP, but not to ACTH or angiotensin II. In addition to expression of steroidogenic enzyme mRNAs, one cell line also expresses mouse renin-1 mRNA, making these cells useful for studies of the role of adrenal renin in regulating adrenal steroidogenesis. These findings represent an approach in transgenic mice to develop highly differentiated adrenal cell lines.
Mol Endocrinol 1994 Jan
PMID:Steroidogenic adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice. 815 34

Steroid 21-hydroxylase activity has been identified in many tissues, including liver. But it is possible that the enzyme found in the liver is different from adrenal 21-hydroxylase. In the adrenal cortex, steroid 21-hydroxylase activity is increased by corticotropin (ACTH); the effect of ACTH is mediated by cyclic AMP (cAMP), and presumably involves a cAMP-dependent protein kinase (PKA). It is not yet clear, however, how extra-adrenal steroid 21-hydroxylase activity is regulated. In the present study, we examined the effect of N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP), forskolin, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (H-8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on steroid 21-hydroxylase activity in primary cultures of rat hepatocytes to determine the nature of regulation of extra-adrenal steroid 21-hydroxylase activity. Steroid 21-hydroxylase activity in hepatocytes incubated with 10(-11) M dbcAMP for 24 h was 1.6 times higher than that in control hepatocytes untreated with dbcAMP. On the other hand, steroid 21-hydroxylase activity decreased by 20 and 50% when the cells were incubated with 10(-5) and 10(-3) M dbcAMP, respectively. The stimulatory effect of 10(-11) M dbcAMP was not blocked by 10(-5) M H-8 (PKA inhibitor), but the inhibitory effect of 10(-5) or 10(-3) M cAMP was. TPA did not alter the activity of steroid 21-hydroxylase. These findings indicate that the steroid 21-hydroxylase in rat liver is regulated by mechanisms different from those in the adrenal glands.
J Steroid Biochem Mol Biol 1994 Apr
PMID:Biphasic regulation by N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) of steroid 21-hydroxylase activity in rat hepatocytes. 818 Jan 19

Congenital adrenal hyperplasia is the most frequent cause of adrenal insufficiency and ambiguous genitalia in newborn children. In contrast to congenital adrenal hyperplasia due to 21-hydroxylase and 11 beta-hydroxylase deficiencies, which impair steroid formation in the adrenal cortex, exclusively, classical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency affects steroid biosynthesis in the gonads as well as in the adrenals. The structures of the highly homologous type I and II 3 beta-HSD genes have been analyzed in three male pseudohermaphrodite 3 beta-HSD deficient patients from unrelated families in order to elucidate the molecular basis of classical 3 beta-HSD deficiency from patients exhibiting various degrees of severity of salt losing. The nucleotide sequence of DNA fragments generated by selective polymerase chain reaction amplification that span the four exons, the exon-intron boundaries, as well as the 5'-flanking region of each of the two 3 beta-HSD genes have been determined in the three male patients. The five point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene, predominantly expressed in the placenta and peripheral tissues. The two male patients suffering from severe salt-losing 3 beta-HSD deficiency are compound heterozygotes, one bearing the frame-shift mutation 186/insC/187 and the missense mutation Y253N, while the other bears the nonsense mutation W171X and the missense mutation E142K. The influence of the detected missense mutations on enzymatic activity was assessed by in vitro expression analysis of mutant recombinant enzymes generated by site-directed mutagenesis in heterologous mammalian cells. Recombinant mutant type II 3 beta-HSD enzymes carrying Y253N or E142K substitutions exhibit no detectable activity. On the other hand, the nonsalt-losing patient is homozygous for the missense mutation A245P. This mutation decreases 3 beta-HSD activity by approximately 90%. The present findings, describing the first missense mutations in the human type II 3 beta-HSD gene, provide unique information on the structure-activity relationships of the 3 beta-HSD superfamily. Moreover, the present findings provide a molecular explanation for the enzymatic heterogeneity responsible for the severe salt-losing form to the clinically inapparent salt-wasting form of classical 3 beta-HSD deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1993 May
PMID:Molecular basis of congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency. 831 54

As part of its trophic action to maintain the steroidogenic capacity of adrenocortical cells, corticotropin (ACTH) increases the transcription of the cytochrome P-450 steroid hydroxylase genes, including the gene encoding steroid 21-hydroxylase (21-OHase). We previously identified several promoter elements that regulate 21-OHase gene expression in mouse Y1 adrenocortical tumor cells. One of these elements, located at nucleotide -65, closely resembles the recognition sequence of the orphan nuclear receptor NGFI-B, suggesting that NGFI-B regulates this essential steroidogenic enzyme. To explore this possibility, we first used in situ hybridization to demonstrate high levels of NGFI-B transcripts in the adrenal cortex of the adult rat. In cultured mouse Y1 adrenocortical cells, treatment with ACTH, the major regulator of 21-OHase transcription, rapidly increased NGFI-B expression. Gel mobility shift and DNase I footprinting experiments showed that recombinantly expressed NGFI-B interacts specifically with the 21-OHase -65 element and identified one complex formed by Y1 extracts and the 21-OHase -65 element that contains NGFI-B. Expression of NGFI-B significantly augmented the activity of the intact 21-OHase promoter, while mutations of the -65 element that abolish NGFI-B binding markedly diminished NGFI-B-mediated transcriptional activation. Specific mutations of NGFI-B shown previously to impair either DNA binding or transcriptional activation diminished the effect of NGFI-B coexpression on 21-OHase expression. Finally, an oligonucleotide containing the NGFI-B response element conferred ACTH response to a core promoter from the prolactin gene, showing that this element is sufficient for ACTH induction. Collectively, these results identify a cellular promoter element that is regulated by NGFI-B and implicate NGFI-B in the transcriptional induction of 21-OHase by ACTH.
Mol Cell Biol 1993 Feb
PMID:The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase. 838 Aug 97

The constitutive and cAMP-induced expression of the mouse steroid 21-hydroxylase gene (Cyp21) are impaired in adrenal cell mutants harboring mutations in cAMP-dependent protein kinase (cAMPdPK). These requirements for a functional cAMPdPK have been mapped to the proximal 330 basepairs of the Cyp21 promoter. This study attempts to identify specific promoter elements of Cyp21 that require cAMPdPK for constitutive activity by comparing their abilities to enhance the expression of a reporter gene in Y1 adrenocortical tumor cells and Y1 Kin mutants defective in cAMPdPK activity. As determined in transient transfection assays, Cyp21 promoter elements at -65, -140, -170, -210, and -280 each enhanced the expression of a human GH reporter gene in parent Y1 cells. The relative order of effectiveness of each of these elements was: -170 >> -280 > -140 > -65 > or = -210. The -170 element was 25-fold more effective in enhancing gene expression from the reporter construct in Y1 cells than in Kin mutant cells; the elements at -65, -140, and -210 were 3-fold more effective in Y1 cells than in Kin mutant cells; the -280 element was equally effective in the parent and Kin mutant clones. These studies suggest that the promoter elements at -170, -65, -140, and -210 mediate the requirement for a functional cAMPdPK in the expression of Cyp21. As determined by gel mobility shift assays with these elements, the dependence of the Cyp21 promoter elements on a functional cAMP-dependent protein kinase did not result from decreased expression or binding affinities of their respective DNA-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1993 Feb
PMID:Identification of promoter elements in the mouse 21-hydroxylase (Cyp21) gene that require a functional cyclic adenosine 3',5'-monophosphate-dependent protein kinase. 838 40

Following three 24 hourly serial injections of 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) to rats, the levels of plasma corticotropin (ACTH) and of adrenal HMG-CoA reductase, the cholesterol side chain cleavage system, 3 beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and adrenodoxin increased after an initial lag of 17 h. In contrast the mRNA level of 11 beta-hydroxylase was differently regulated since it was elevated after 17 and 24 h and decreased thereafter to basal values. These increases appear to be related to ACTH secretion since they were blocked by the coadministration of dexamethasone (Dex) and 4-APP. Also 3 h after the administration of Dex to 4-APP treated rats rapid decreases in plasma corticosterone and ACTH levels were accompanied by decreases in mRNA levels of HMG-CoA reductase and low density lipoprotein receptor, two components involved in the synthesis and transport of cholesterol. The mRNA level of the electron donor adrenodoxin was also decreased, suggesting that this component participates in the short term regulation of corticosterone synthesis in the rat adrenal. The adrenal response was more readily observed with components involved in the steps preceding cholesterol biosynthesis than in those subsequent to cholesterol in the corticosteroid pathway. However, the effects of 4-APP on the latter pathway were well documented with mRNA analysis performed by Northern blot, a more sensitive technique than the Western blot used for protein quantification. The entire metabolism of the corticosterone biosynthetic pathway was thus affected in rats treated with 4-APP. Taken collectively these results indicate that under acute lipoprotein depletion rat adrenals developed a compensatory mechanism enabling them to synthesize and utilize cholesterol for corticosteroid synthesis.
Mol Cell Endocrinol 1993 May
PMID:Effects of dexamethasone on the levels of adrenal steroidogenic enzyme mRNA in rats treated with 4-aminopyrazolopyrimidine. 839 95

The cytochrome P450 steroid hydroxylases are coordinately regulated by steroidogenic factor 1 (SF-1), a protein expressed selectively in steroidogenic cells. Based on its expression in steroidogenic tissues and DNA-binding specificity, we isolated a putative SF-1 cDNA from an adrenocortical cDNA library. As evidence that this cDNA encodes SF-1, we now show that it is selectively expressed in steroidogenic cells, that an antiserum against its protein product specifically abolishes the SF-1-related gel-shift complex, and that its coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. Sequence analyses of the SF-1 cDNA revealed that it is the mouse homolog of fushi tarazu factor I (FTZ-F1), a nuclear receptor that regulates the fushi tarazu homeobox gene in Drosophila. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contained both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defined shared exons encoding common regions and alternative promoters and 3'-exons leading to differences between the two FTZ-F1 transcripts. We used in situ hybridization with transcript-specific probes to study the ontogeny of SF-1 and ELP expression. ELP transcripts were not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1993 Jul
PMID:Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. 841 9

A number of biochemical tests have been utilized to assist the diagnosis of steroid 21-hydroxylase deficiency. The specificity and accuracy of plasma 17-hydroxyprogesterone assays are important. A profile of steroids in urine by gas chromatography and mass spectrometry is the definitive test. Molecular biology is not practical for the diagnosis of a new case. The ACTH stimulation test for detection of heterozygotes is a poor discriminant. Fertility in patients with congenital adrenal hyperplasia may be due to excess of progesterone as well as of androgens. Gene amplification offers the best approach in molecular biology for the prenatal diagnosis of 21-hydroxylase deficiency.
J Steroid Biochem Mol Biol 1993 Apr
PMID:Problems in diagnosis and management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. 848 53


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