Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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An enzyme-mediated metabolism of androgens and estrogens including 17beta-HSD activity in the brain of vertebrates was discovered approximately 30 years ago. Mainly 5alpha-reductase and aromatase have been studied in detail. Recently we could demonstrate reductive and oxidative 17beta-HSD activity as well as considerable mRNA expression of the 17beta-HSD types 3 and 4 in the human brain. In the present study, we report on 17beta-HSD type 5 mRNA expression in brain tissue of women and men. Data analysis did not reveal sex specific differences, but we determined a significantly higher mRNA concentration in the subcortical white matter (SC) than in the cerebral cortex (CX). Investigation of reductive 17beta-HSD in vitro activity with 2 microM androstenedione as the substrate revealed no sex specific differences. Testosterone formation was significantly higher in SC than in CX. Moreover, enzyme activity was significantly higher in brain tissue of adults compared to that of children.
Mol Cell Endocrinol 2001 Jan 22
PMID:Expression of the 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the human brain. 1116 25

The relative abundance and physiological role of 5alpha-reductase (5alphaR) isoforms in rat testis, in particular 5alpha-reductase Type 2 (5alphaR2) are poorly understood. Investigation of 5alphaR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5alpha-reductase Type 1 (5alphaR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5alphaR assays measured the conversion of 3[H]-testosterone to 5alpha-reduced metabolites (dihydrotestosterone+3alpha-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5alphaR1 activity at pH 5.0, the amount of 5alphaR1 activity at pH 5.0 was determined by measuring recombinant rat 5alphaR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5alphaR1 was determined to be 12.4+/-1.4% (mean+/-S.D., n=14). The 5alphaR2 assay was validated by determining recombinant rat 5alphaR2 activity in the presence of recombinant rat 5alphaR1 activity in COS cells. A 99.3+/-14.7% recovery of 5alphaR2 activity was obtained when comparing 5alphaR2 activity recovered versus activity added. 5alphaR1 and 5alphaR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50-100-fold) over this age range, with 5alphaR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5alphaR2 activity in the presence of high concentrations of 5alphaR1 was developed and is applicable in the measurement of 5alphaR2 activity in rat testis.
J Steroid Biochem Mol Biol 2000 Dec 01
PMID:Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis. 1117 11

Impaired virilisation during embryonic development and pubertal arrest in patients with androgen insensitivity syndromes (AIS) is usually caused by mutations in the androgen receptor (AR)- or the 5alpha-reductase II (5RII) gene. However, identical mutations may lead to strikingly different phenotypes. To investigate whether this may be caused by individually altered transcription rates in fibroblasts from the genital region (GF) from affected patients, we applied competitive reverse transcribed PCRs (competitive RT-PCR) targeting AR- and 5RII-transcripts. We could demonstrate that AR- and 5RII-mRNA concentrations in cells from patients with partial and complete AIS and missense mutations in the AR- or 5RII-gene are normal or only moderately lowered compared to equally aged normal controls. However, in a patient bearing a premature stop-codon in the AR-gene a considerably lowered AR-transcript level was detected. We conclude, that in patients with incomplete virilisation disorders due to missense mutations, transcription regulation of AR and 5RII generally follows normal patterns. Accordingly, the premature stop-codon found in one patient's AR-gene may rather cause reduced transcript stability than an impairment of transcription activity. Therefore, altered AR- and 5RII-transcription rates in fibroblasts from the GF do not seem to be the cause for the variable genotype-phenotype correlation in androgen insensitivity syndrome.
J Steroid Biochem Mol Biol 2000 Dec 31
PMID:Transcription of androgen receptor and 5alpha-reductase II in genital fibroblasts from patients with androgen insensitivity syndrome. 1128 74

Site directed mutagenesis of human steroid 5alpha-reductase types 1 (5AR1) and 2 (5AR2) has been used to identify residues involved in inhibitor/substrate binding by 5AR2. Replacing residues 21-24 (GALA) in 5AR2 with the analogous residues 26-29 (AVFA) from 5AR1 did not significantly alter either the Km for testosterone or the Ki for the competitive inhibitor Finasteride. Replacement of AVFA in 5AR1 with GALA from 5AR2 however, significantly decreased the Km and increased the resistance to Finasteride. These findings confirm that 5AR1 residues 26-29 are involved in inhibitor/substrate binding but suggest residues 21-24 of 5AR2 are not. Replacing residues 20-29 (QCAVGCAVFA) of 5AR1 with the analogous residues 15-24 (ATLVALGALA) from 5AR2, changed the Km and Ki to values approaching those for wild type 5AR2. Replacing residues VAL in wild type 5AR2 with VGC from 5AR1 did not change Km or Ki but replacing ATL in 5AR2 with QCA from 5AR1 significantly decreased the Km and increased the resistance to Finasteride. Conversely, replacing QCA with ATL in 5AR1 containing GALA in place of AVFA, increased the Km and decreased resistance to Finasteride. These findings indicate residues 15-17 of human 5AR2 participate in inhibitor/substrate binding whereas residues 18-20 do not.
J Steroid Biochem Mol Biol 2001 May
PMID:Structure-function studies of human 5-alpha reductase type 2 using site directed mutagenesis. 1137 83

We have investigated the roles of highly conserved glycine (G175, G185), negatively charged (E188, D165) and histidine residues (H233, H237) in rat steroid 5alpha-reductase (isozyme-1), on NADPH, testosterone (T) binding and enzyme activity. The mutations G175R and G175S result in a two- to threefold increase in K(m)(NADPH) and an approximately fourfold decrease in the V(max) with no change in K(m)(T). The mutation G185W resulted in a fivefold decrease in K(m)(NADPH) and an eightfold decrease in V(max), with no change in K(m)(T), whereas the mutations E188Q and D165N both resulted in inactive enzyme. Steady-state kinetic measurements showed that the mutation H233R resulted in an approximately 40-fold decrease in V(max), an approximately 20-fold increase in K(m)(T) and no alteration in K(m)(NADPH), whereas the mutation H237R resulted in virtually inactive enzyme. The results suggest that the conserved glycines are not essential for cofactor binding and activity, and that the negatively charged residues may contribute to enzyme stability, whereas the C-terminal histidines appear to be involved in substrate binding and catalytic activity.
J Steroid Biochem Mol Biol 2001 May
PMID:Site-directed mutagenesis studies of rat steroid 5alpha-reductase (isozyme-1): mutation of residues in the cofactor binding and C-terminal regions. 1137 84

Inhibitors of human 5alpha-reductase type II are promising drug candidates for the treatment of benign prostatic hyperplasia which is associated with high prostatic DHT levels. In this study we describe the evaluation of potential inhibitors in a new cell assay. First a plasmid (pRcCMV-5alphaII) for the expression of human 5alpha-reductase type II was constructed by the use of the vector pRcCMV and transfected into the African green monkey fibroblast-like cell line COS1. By selection with G418 sulfate, ten COS1 single cell clones were obtained of which three stably exhibited high 5alpha-reductase activity. One single cell clone (COS1-5alphaIIST) was selected for further investigations. By Southern blot analysis, fluorescence in situ hybridization (FISH) and comparative PCR experiments it turned out that the expression plasmid pRcCMV-5alphaII has been integrated into the chromosome, resulting in a long-term stable expression of the foreign 5alpha-reductase gene. The newly established cell line was used for testing novel compounds on their inhibitory effect on human 5alpha-reductase type II. Using this whole cell assay, inhibitors with IC(50) values in the nanomolar range could be identified.
J Steroid Biochem Mol Biol 2001 Sep
PMID:Stable expression of human 5alpha-reductase type II in COS1 cells due to chromosomal gene integration: a novel tool for inhibitor identification. 1159 8

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.
J Steroid Biochem Mol Biol 2002 Mar
PMID:Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes. 1194 17

4-Tert-octylphenol (OP) is a breakdown product of 4-tert-octylphenol ethoxylate, which is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to exhibit weak estrogenic activity in many assay systems. The studies described herein examined an unusual effect of OP in increasing constitutive testosterone levels of cultured Leydig cells from young adult rats. The increase in testosterone was both dose and time sensitive, and this response was observed in medium lacking both calcium and magnesium and containing a membrane-permeable calcium chelator, suggesting that the increase in testosterone was not mediated by an increase in the permeability of extracellular calcium into cells or the redistribution/release of calcium from intracellular stores, respectively. Cellular cAMP levels also were unaffected by OP alone in cultured Leydig cells. Furthermore, initial exposure to 2000nM OP alone for 4h did not alter the subsequent conversion of endogenous cholesterol or exogenously added 22 (R)hydroxycholesterol to testosterone, suggesting that the increase in testosterone was not due to the enhanced availability of endogenous cholesterol or an increase in cholesterol side-chain cleavage activity, respectively. The increase in testosterone also was observed in the presence of the pure estrogen antagonist, ICI 182,780, or a 5alpha-reductase inhibitor, suggesting that this effect of OP was not mediated through the estrogen receptor alpha or beta pathway or by inhibition of Leydig cell testosterone metabolism, respectively. In addition, exposure of cells to comparable concentrations of two different detergents, Triton X-100 or sodium cholate, did not increase testosterone levels, suggesting that this effect of OP was not due to its potential detergent qualities. Although these studies did not identify specific mechanism(s) that increase constitutive testosterone levels by OP, they identify specific pathways that appear not to be involved. The physiological relevance of this observation is not known; nevertheless, they illustrate potential diverse actions of OP in modulating the level of androgen secreted by Leydig cells, and they emphasize that some actions of OP do not appear to be mediated through the estrogen receptor alpha or beta pathway.
J Steroid Biochem Mol Biol 2002 Jun
PMID:Exposure to octylphenol increases basal testosterone formation by cultured adult rat Leydig cells. 1213 9

Patients with Smith-Lemli-Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-delta(7)-reductase which catalyses the final step in cholesterol synthesis. Accumulation of 7- and 8-dehydrocholesterol is a result of the disorder and potentially these sterols could be used as precursors of a novel class of delta(7) and delta(8) unsaturated adrenal steroids and their metabolites. In this study, we have analyzed urine from SLOS patients in the anticipation of characterizing such metabolites. Gas chromatography/mass spectrometry (GC/MS) was used in the identification of two major metabolites as 7- and 8-dehydroversions of the well-known steroid pregnanetriol. Other steroids, such as 8-dehydro dehydroepiandrosterone (8-dehydro DHEA) and 7- or 8-dehydroandrostenediol were also identified, and several more steroids are present in urine but remain uncharacterized. As yet, the study provides no evidence for the production of ring-B unsaturated metabolites of complex steroids, such as cortisol. We believe that the following transformations can utilize ring-B dehydroprecursors: StAR transport of cholesterol, p450 side chain cleavage, 17-hydroxylase/17,20-lyase, 3beta-hydroxysteroid dehydrogenase, 3alpha-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 5beta-reductase. We have yet to prove the activity of adrenal 21-hydroxylase, 11beta-hydroxylase or 5alpha-reductase towards 7- or 8-dehydroprecursors.
J Steroid Biochem Mol Biol 2002 Oct
PMID:Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-delta7-reductase deficiency (Smith-Lemli-Opitz syndrome). 1247 89

In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%. To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector. The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml). The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.
J Steroid Biochem Mol Biol 2002 Oct
PMID:Inhibition of type 1 and type 2 5alpha-reductase activity by free fatty acids, active ingredients of Permixon. 1247 90


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