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I Dufort

Publications and source records attributed to I Dufort.

14 recordsLinked to original sources
PubMed2001

Type 5 17beta-hydroxysteroid dehydrogenase: its role in the formation of androgens in women.

Type 5 17beta-HSD, one of the seven types of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) so far characterized in humans, catalyzes the transformation of 4-androstenedione (4-dione) into testosterone (T). This reaction is also catalyzed by type 3 17beta-HSD which is responsible for pseudohermaphroditism in deficient man but is asymptomatic in deficient women. Since type 3 17beta-HSD is not found in the ovary, whereas type 5 is, it is suggested that the latter is involved in the conversion of 4-androstenedione to testosterone in the ovary. The comparison of type 5 17beta-HSD of different species shows that the human enzyme shares 95 and 78% identity with the monkey and mouse enzymes respectively. In addition, the human and monkey enzymes are labile and are destroyed upon homogenization of the transfected cells, whereas the mouse enzyme is not. Human type 5 17beta-HSD also possesses a high 20alpha-HSD activity that inactivates progesterone, whereas the monkey and mouse enzymes do not have such high 20alpha-HSD activity. Since the endocrine ovary is composed of two types of cells, one producing androgens (theca cells) and the other producing progesterone and estrogens (granulosa cells), it is tempting to suggest that the role of the high 20alpha-HSD activity of type 5 17beta-HSD is to protect the theca cells against the progesterone produced by the granulosa cells. The double activity of type 5 17beta-HSD in the female reproductive tissues is probably necessary to the control of the optimal level of progesterone and sex steroids.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

High metabolization of catecholestrogens by type 1 estrogen sulfotransferase (hEST1).

Recently, two types of estrogen sulfotransferase, chronologically named types 1 and 2 estrogen sulfotransferase (hEST1 and hEST2), have been described. Since hEST2 selectively catalyzes the sulfonation of ethinyl estradiol as well as that of estrone (E1) and estradiol (E2), but poorly the sulfonation of catecholestrogens, we wanted to assess the ability of hEST1 to metabolize these compounds. We overexpressed hEST1 in Escherichia coli in fusion with GST, then purified the enzyme using a glutathione affinity column, and obtained GST-free enzyme by digestion with thrombin. Using [35S]-phosphosadenosine phosphosulfate (PAPS) as cofactor, we showed that hEST1 efficiently metabolizes the transformation of 2-OH-E2 and 2-OH-E1. However, the transformation of 4-OH-E1 and 4-OH-E2 is much less efficient. Our results also show that hEST1 metabolizes more efficiently E2 than E1. Since hEST1 mRNA is produced from the same gene as MPST using different alternative promoters and since it is expressed in most breast cancer cells (MCF-7, ZR-75-1, T47-D, MDA-231, and MDA-418), studies of the expression and activity of hEST1 will be most important to have a better knowledge about its involvement in the control of the genotoxicity of estrogens and catecholestrogens.

Base Sequence↗
PubMed2001

Comparative biosynthetic pathway of androstenol and androgens.

It has been shown recently that androstenol and androstanol could modulate gene expression through the nuclear orphan receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor). Although, in the pig, androstenol is produced in high amounts and is active as a pheromone, its role in the human is ill defined. Androstenol possesses a structure similar to that of androgens, with the exception that it does not possess an oxygen at position 17 that is crucial for androgenic and estrogenic activity. It has been shown that human and boar testis homogenates could produce androstenol, but details of the biosynthetic pathway had not yet been elucidated. It has also been shown recently that androstenol could modulate the activity of CAR and PXR and the expression of some cytochrome P450 drug-metabolizing enzymes. We wanted to determine the precise biosynthetic pathway of androstenol and other closely related steroids. Using transformed human embryonic kidney (HEK-293) cells that stably express 3 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, we have shown that these enzymes are able to efficiently transform the precursor 5,16-androstadien-3 beta-ol into androstenol. We thus provided evidence that androstenol, the ligand for CAR and PXR, is produced by the biosynthetic pathway of sex steroids.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

Human types 1 and 3 3 alpha-hydroxysteroid dehydrogenases: differential lability and tissue distribution.

3 alpha-Hydroxysteroid dehydrogenases (3 alpha-HSDs) catalyze the conversion of 3-ketosteroids to 3 alpha-hydroxy compounds. The best known 3 alpha-HSD activity is the transformation of the most potent natural androgen, dihydrotestosterone, into 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), a compound having much lower activity. Previous reports show that 3 alpha-HSDs are involved in the metabolism of glucocorticoids, progestins, prostaglandins, bile acid precursors, and xenobiotics. 3 alpha-HSDs could, thus, play a crucial role in the control of a series of active steroid levels in target tissues. In the human, type 1 3 alpha-HSD was first identified as human chlordecone reductase. Recently, we have isolated and characterized type 3 3 alpha-HSD that shares 81.7% identity with human type 1 3 alpha-HSD. The transfection of vectors expressing types 1 and 3 3 alpha-HSD in transformed human embryonic kidney (HEK-293) cells indicates that both enzymes efficiently catalyze the transformation of dihydrotestosterone into 3 alpha-diol in intact cells. However, when the cells are broken, the activity of type 3 3 alpha-HSD is rapidly lost, whereas the type 1 3 alpha-HSD activity remains stable. We have previously found that human type 5 17 beta-HSD which possesses 84% and 86% identity with types 1 and 3 3 alpha-HSD, respectively, is also labile, whereas rodent enzymes such as mouse type 5 17 beta-HSD and rat 3 alpha-HSD are stable after homogenization of the cells. The variable stability of different enzymatic activities in broken cell preparations renders the comparison of different enzymes difficult. RNA expression analysis indicates that human type 1 3 alpha-HSD is expressed exclusively in the liver, whereas type 3 is more widely expressed and is found in the liver, adrenal, testis, brain, prostate, and HaCaT keratinocytes. Based on enzymatic characteristics and sequence homology, it is suggested that type 1 3 alpha-HSD is an ortholog of rat 3 alpha-HSD while type 3 3 alpha-HSD, which must have diverged recently, seems unique to human and is probably more involved in intracrine activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Characterization of a human 20alpha-hydroxysteroid dehydrogenase.

It has been suggested that 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) is a T-cell differentiation marker in mice. In the human, this enzyme has generally been associated with types 1 and 2 17beta-HSDs, which belong to the short-chain alcohol dehydrogenase family, whereas the rat, rabbit, pig and bovine 20alpha-HSDs are members of the aldoketo reductase superfamily, which also includes the 3alpha-HSD family. In this study, we report the cloning, from a human skin cDNA library, of a cDNA that shows, after transfection into human embryonic kidney (HEK-293) cells, high 20alpha-HSD activity but negligible 3alpha- and 17beta-hydroxysteroid dehydrogenase activities. A comparison of the amino acid sequence of the human 20alpha-HSD with those of other related 20alpha- and 3alpha-HSDs indicates that the human 20alpha-HSD shares 79.9, 68.7 and 52.3% identity with rabbit, rat and bovine 20alpha-HSDs, whereas it shows 97, 84 and 65% identity with human type 3, type 1 and rat 3alpha-HSDs. In contrast, the enzyme shares only 15.2 and 15.0% identity with type 1 and type 2 human 17beta-HSDs. DNA analysis predicts a protein of 323 amino acids, with a calculated molecular weight of 36 767 Da. In intact transfected cells, the human 20alpha-HSD preferentially catalyzes the reduction of progesterone to 20alpha-hydroxyprogesterone with a K(m) value of 0.6 microM, the reverse reaction (oxidation) being negligible. In a cell cytosolic preparation, the enzyme could use both NADPH and NADH as cofactors, but NADPH, which gave 4-fold lower K(m) values, was preferred. We detected the expression of 20alpha-HSD mRNA in liver, prostate, testis, adrenal, brain, uterus and mammary-gland tissues and in human keratinocyte (HaCaT) cells. The present study clearly indicates that the genuine human 20alpha-HSD belongs to the aldoketo reductase family, like the 20alpha-HSDs from other species.

20-Hydroxysteroid Dehydrogenases↗
PubMed1999

Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase.

17Beta-hydroxysteroid dehydrogenases (17betaHSDs) play an essential role in the formation of active intracellular sex steroids. Six types of 17betaHSD have been described to date, which only share approximately 20% homology. Human type 5 17betaHSD complementary DNA is unique among the 17betaHSDs because it belongs to the aldo-keto reductase family, whereas the others are members of the short chain alcohol dehydrogenases. The characteristics of human type 5 17betaHSD were investigated in human embryonic (293) cells stably transfected with human and mouse type 5 17betaHSD, as well as human type 3 3alphaHSD. Using intact transfected cells, type 5 17betaHSD shows a substrate specificity pattern comparable to those of human type 3 17betaHSD and mouse type 5 17betaHSD. These enzymes catalyze more efficiently the transformation of androstenedione (4-dione) to testosterone, whereas the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol is much lower. In contrast, type 3 3alphaHSD catalyzes more efficiently the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol, whereas the transformation of 4-dione to testosterone represents only 7% of the 3alphaHSD activity. However, upon homogenization, human type 5 17betaHSD activity decreases to approximately 10% of the activity in intact cells and remains stable at this level together with the 3alphaHSD activity. Under the same conditions, however, the mouse enzyme is not altered by homogenization. Indeed, using purified human 17betaHSD overexpressed in Escherichia coli, we could confirm that a much greater amount of protein is required to produce activity similar to the enzymatic activity measured in intact transfected cells. The present data provide the answer to the question of why previous researchers could hardly detect type 5 17betaHSD activity. Indeed, all previous publications used cell or tissue homogenates or purified enzymes. Under such conditions, only the low level, but stable, 3alphaHSD and 17betaHSD activities could be measured, whereas the high level, but highly unstable, 17betaHSD activity could not be measured. As type 5 17betaHSD shares 84%, 86%, and 88% amino acid identity with types 1 and 3 3alphaHSD and 20alphaHSDs, respectively, Northern blot analysis used in previous studies could not provide unequivocal information. In this report, we used a more specific ribonuclease protection assay and could thus show that human type 5 17betaHSD is expressed in the liver, adrenal, and prostate; in prostatic cancer cell lines DU-145 and LNCaP; as well as in bone carcinoma (MG-63) cells. By analogy with type 3 17betaHSD, which is responsible for the formation of androgens in the testis, the expression of type 5 17betaHSD in the prostate and bone cells suggests that this enzyme is involved in the formation of active intracellular androgens in these tissues.

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

Molecular cloning of human type 3 3 alpha-hydroxysteroid dehydrogenase that differs from 20 alpha-hydroxysteroid dehydrogenase by seven amino acids.

We have isolated, by screening a lambda gt11 human prostatic cDNA library, a cDNA clone that shows after transfection into transformed human embryonal kidney (293) cells high 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity that catalyzes efficiently the transformation of dihydrotestosterone to 5 alpha-androstane-3 alpha, 17 beta-diol. Chronologically, we name this enzyme type 3 3 alpha-HSD (3 alpha-HSD3). Surprisingly, human 3 alpha-HSD3 shares much higher amino acids sequence identity with human 20 alpha-HSD (97.8%) than with human type 1 and type 2 3 alpha-HSD (81.1 and 85.7% identity, respectively). DNA analysis predicts a protein of 323 amino acids with a molecular mass of 36,844. Alignment of the amino acid sequence of 3 alpha-HSD3 with other related 3 alpha- and 20 alpha-HSDs indicates that 3 alpha-HSD3 shares 68.1, 78.3, and 67.4% identity with rat 3 alpha-HSD and rabbit and rat 20 alpha-HSD, respectively. 3 alpha-HSD3 belongs to the aldo-keto reductase family and like almost all the members of this family preferred NADPH as cofactor.

20-Hydroxysteroid Dehydrogenases↗
PubMed1996

Isolation and characterization of a stereospecific 3beta-hydroxysteriod sulfotransferase (pregnenolone sulfotransferase) cDNA.

In contrast to humans, who possess a hydroxysteroid sulfotransferase (HSST), namely, DHEA sulfotransferase (DHEA-ST), that displays broad substrate specificities, HSSTs of the guinea pig show a high substrate stereoselectivity, as shown by the recent cloning of a chiral-specific 3alpha-hydroxysteroid sulfotransferase. Herein, we report the cloning and expression of the substrate and chiral-specific pregnenolone sulfotransferase (PREG-ST). Transfection of the pCMV expression vector containing PREG-ST cDNA in transformed human embryonal kidney (293) cells showed that the expressed enzyme selectively catalyzes the 3beta-hydroxysteroid substrate. It converts pregnenolone to pregnenolone sulfate most efficiently, whereas dehydroepiandrosterone and epiandrosterone were transformed at a much lower rate, and androsterone, a 3alpha-hydroxysteroid, was not significantly metabolized (30-fold lower). Thus, the enzyme was identified as pregnenolone sulfotransferase. DNA analysis predicts a protein of 287 amino acids with a calculated molecular mass of 34,199 daltons. Alignment of the amino acid sequence with other sulfotransferases indicated that guinea pig pregnenolone sulfotransferase shares 75 and 80% homology with human DHEA sulfotransferase and rat hydroxysteroid dehydrogenase, respectively. RNA blot analysis using guinea pig liver, intestine, adrenal, kidney, epididymis, testis, and lung showed a single RNA species at 1.3 kb is expressed in liver, intestine, and kidney. Guinea pig 3beta-hydroxysteroid sulfotransferase is thus different from that in humans, who possess two mRNA species of 1.3 and 1.8 kb.

Amino Acid Sequence↗
PubMed1996

Steroid sulfotransferases.

Human dehydroepiandrosterone sulfotransferase (DHEA-ST) catalyzes the sulfonation of DHEA, cholesterol, pregnenolone as well as androsterone. RNA blot analysis shows two DHEA-ST mRNA species of 1.3 and 1.8 kb that are expressed similarly in liver and adrenals. To determine whether the form expressed in adrenals is distinct or identical with the one expressed in liver, we have cloned and sequenced the 1.8 kb DHEA-ST cDNA from human adrenal cDNA library. Except for one nucleotide difference, the human adrenal and liver DHEA-ST cDNAs are identical. Using expression vectors containing the chloramphenicol acetyltransferase (CAT) reporter gene ligated to various fragments of the DHEA-ST gene promoter, we have shown that DHEA-ST gene promoter activity is stimulated by estradiol (E2). The E2 stimulation is inhibited by the anti-estrogen EM-139. In contrast to human DHEA-ST, guinea pig hydroxysteroid sulfotransferases show high substrate- and stereo-selectivity. We have cloned a chiral-specific pregnenolone sulfotransferase (PREG-ST) which catalyzes mainly the transformation of pregnenolone to pregnenolone sulfate. Estrogen sulfotransferase catalyzes the conversion of estrone and estradiol to their inactive sulfated forms and could thus play a major role in the control of estrogen levels in target tissues. Recently, using a probe derived from bovine estrogen sulfotransferase, we have cloned a cDNA and gene that we first named human estrogen sulfotransferase (hEST) since the expressed enzyme is able to transform estrone to estrone sulfate. Actually, the Hugo nomenclature committee named this gene STM gene because it also codes for monoamine-sulfating phenol-sulfotransferase (M-PST). hEST1 possesses the same coding and 3'-untranslated region as human brain aryl sulfotransferase (HAST) and M-PST, but different 5'-noncoding region. Analysis of hEST1 gene sequence indicates that hEST1 and HAST3 or M-PST mRNA species are transcribed from a single hEST1 gene by alternative promoters using two separate exon 1, named exon Ia and exon Ib. We also described the identification of a third mRNA species (M-PST gamma) issued from the STM gene and the characterization of the structure of the phenol-sulfating phenolsulfotransferase (STP) gene that is highly homologous to the STM gene. Similar to STM, the STP gene generates multiple mRNA species that differ only in the 5'-untranslated sequence.

Adrenal Glands↗
PubMed1995

Genetic linkage mapping of the dehydroepiandrosterone sulfotransferase (STD) gene on the chromosome 19q13.3 region.

In the human liver and adrenal, there is a single hydroxysteroid sulfotransferase, which catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundantly circulating steroid in humans, and also catalyzes the sulfation of a series of other 3 beta-hydroxysteroids as well as cholesterol. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in several peripheral tissues, indicating that hydroxysteroid sulfotransferase plays a pivotal role in controlling the hormonal action of sex steroids by regulating their bioavailability. We recently elucidated the structure of the gene encoding hydroxysteroid sulfotransferase (STD), also designated dehydroepiandrosterone sulfotransferase, which spans 17 kb and contains six exons. The STD gene was preliminarily assigned to chromosome 19 by polymerase chain reaction (PCR) amplification of DNA from a panel of human/rodent somatic cell hybrids. To locate the STD gene, the novel biallelic polymorphism found in intron 2 was genotyped in eight CEPH reference families by direct sequencing of PCR products. Two-point linkage analysis was first performed between the latter polymorphism and chromosome 19 markers from Généthon and NIH/CEPH. The closest linkage was observed with D19S412 (Zmax = 9.23; theta max 0.038) and HRC (Zmax = 5.95; theta max 0.036), located on the 19q13.3 region. A framework map including six Généthon markers flanking the polymorphic STD gene was created by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the region was constructed, yielding to the following order: qter-D19S414-D19S224-D19S420-D19S217-(APOC2++ +-D19S412)- (STD-HRC)-KLK-D19S22-D19S180-PRKCG-D19S418 -tel.

Base Sequence↗
PubMed1995

Structural characterization and expression of the human dehydroepiandrosterone sulfotransferase gene.

Dehydroepiandrosterone sulfotransferase catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundant steroid in circulation in the human and primate. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in peripheral target tissues. In addition, blockade at the dehydroepiandrosterone level could give raise to high level of DHEA and thus disorders due to mild excess of androgen. Recently, the cDNA encoding dehydroepiandrosterone sulfotransferase has been isolated from a human liver cDNA library. To study the regulation and expression, as well as the possible defect linked to DHEA sulfotransferase gene, we have isolated and characterized its structure by screening a lambda EMBL3 library of human leukocyte genomic DNA using human dehydroepiandrosterone sulfotransferase cDNA as a probe. Sequencing of the gene shows that it is included in approximately 17 kb and contains six exons separated by five introns. Northern blot analysis shows a strong signal in the adrenals and liver, whereas no signal was detected in the spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, lung, skeletal muscle, kidney, or pancreas. Using primer extension analysis, the transcription start site is located at nucleotide 98 upstream from the ATG initiating codon. Putative TATA and CAAT boxes are situated at positions 72 and 96 upstream from the transcription start site, respectively. Using DNA from a panel of human/rodent somatic cell hybrids, and amplification of the gene by the polymerase chain reaction, the human dehydroepiandrosterone sulfotransferase gene has been assigned to chromosome 19.

Amino Acid Sequence↗