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.     

Clinical, endocrine, and molecular findings in 17beta-hydroxysteroid dehydrogenase type 3 deficiency

The 17beta-hydroxysteroid dehydrogenases (17betaHSD) gene family comprises different enzymes involved in the biosynthesis of active steroid hormones. The 17betaHSD type 3 (17betaHSD3) isoenzyme catalyzes the reductive conversion of the inactive C19-steroid, Delta4-androstenedione (Delta4- A), into the biologically active androgen, testosterone (T), in the Leydig cells of the testis. It is encoded by the 17beta-hydroxysteroid dehydrogenase type 3 (HSD17B3) gene, which maps to chromosome 9q22. Mutations in the HSD17B3 gene are associated with a rare form of 46,XY disorder of sex development referred to as 17betaHSD3 deficiency (or as 17-ketosteroid reductase deficiency), due to impaired testicular conversion of Delta4-A into T. 46,XY patients with 17betaHSD3 deficiency are usually classified as female at birth, raised as such, but develop secondary male features at puberty. Diagnosis, and consequently early treatment, is difficult because clinical signs from birth until puberty may be mild or absent. Biochemical diagnosis of 17betaHSD3 deficiency requires measurement of serum T/Delta4-A ratio after hCG stimulation test in pre-pubertal subjects, while baseline values seem to be informative in early infancy and adolescence. However, low basal T/Delta4-A ratio is not specific for 17betaHSD3 deficiency, being sometimes also found in patients with other defects in T synthesis or with Leydig cells hypoplasia. Mutational analysis of the 17HSDB3 gene is useful in confirming the clinical diagnosis of 17betaHSD3 deficiency. This review describes clinical findings, diagnosis, and molecular basis of this rare disease.     

Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase.

I have isolated glucose-6-phosphate dehydrogenase from rabbit liver microsomes and determined its complete amino acid sequence. Sequence determination was achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The microsomal enzyme consists of 763 residues and is quite dissimilar from the previously characterized cytosolic enzymes. The N terminus of the microsomal enzyme is blocked by a pyroglutamyl residue. Carbohydrate is attached at Asn-138 and Asn-263, implying that the bulk of the protein is oriented on the lumenal side of the endoplasmic membrane. The amino acid sequence of the microsomal protein shows limited homology to the extensively sequenced cytosolic glucose-6-phosphate dehydrogenases. Clusters of up to six identical residues can be identified in four regions: peptide segments at residues 10-21, 154-163, and 173-261. In addition, another array of identical residues, requiring a 100-residue deletion in the sequence of the microsomal enzyme, spans residues 436-462 and corresponds to residues 348-373 of the cytosolic protein. Two segments with a Gly-Xaa-Gly-Xaa-Xaa-Gly motif, related to a coenzyme binding fold, were identified at Gly-399 and Gly-491. In the cytosolic enzymes, a variation of this sequence motif occurs at Gly-37 and Gly-241. The 300-residue C-terminal segment of the microsomal enzyme is unique and has no counterpart in the cytosolic or the bacterial enzymes. An unexpected finding with regard to the microsomal enzyme is that it lacks an identifiable membrane-spanning region or the lumenal-protein C-terminal consensus sequences Lys-Asp-Glu or His-Ile/Thr-Glu-Leu. Thus, the mode of transport and retention of this protein in the lumen of endoplasmic reticulum remains to be determined.     

Molecular characterization of two plant flavonol sulfotransferases.

cDNA clones coding for flavonol 3- and 4'-sulfotransferases (STs) were isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA extracted from terminal buds of Flaveria chloraefolia. Sequence analysis revealed full-length cDNA clones with open reading frames of 933 and 960 base pairs, which encode polypeptides containing 311 and 320 amino acids, respectively. This corresponds to a molecular mass of 36,442 Da for the 3-ST and 37,212 Da for the 4'-ST. Expression of these clones in Escherichia coli led to the synthesis of beta-galactosidase-ST fusion proteins having the same substrate and position specificities as those for the 3- and 4'-flavonol ST enzymes isolated from the plant. Comparison of the deduced amino acid sequence of the two clones revealed an overall identity of 69% in 311 amino acid residues. The two flavonol STs of F. chloraefolia also shared significant sequence similarities with steroid and aryl STs found in animal tissues and with the senescence marker protein 2 isolated from rat liver, suggesting an evolutionary link between plant and animal STs.     

Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase.

The molecular structure of a rat hepatoma 70-kDa insulin/mitogen-stimulated S6 protein kinase, obtained by molecular cloning, is compared to that of a rat homolog of the 85-kDa Xenopus S6 protein kinase alpha; both kinases were cloned from H4 hepatoma cDNA libraries. The 70-kDa S6 kinase (calculated molecular mass of 59,186 Da) exhibits a single catalytic domain that is most closely related in amino acid sequence (56% identity) to the amino-terminal, kinase C-like domain of the rat p85 S6 kinase (calculated molecular mass of 82,695 Da); strong similarity extends through a further 67 residues carboxyl-terminal to the catalytic domain (40% identity), corresponding to a region also conserved among the kinase C family. Outside of this segment of approximately 330 amino acids, the structures of the p70 and p85 S6 kinases diverge substantially. The p70 S6 kinase is known to be activated through serine/threonine phosphorylation by unidentified insulin/mitogen-activated protein kinases. A model for the regulation of p70 S6 protein kinase activity is proposed wherein the low activity of the unphosphorylated enzyme results from the binding of a basic, inhibitory pseudosubstrate site (located carboxyl-terminal to the extended catalytic domain) to an acidic substrate binding region (located amino-terminal to the catalytic domain); substrate binding is thereby prevented. S6 kinase activation requires displacement of this inhibitory segment, which is proposed to occur consequent to its multiple phosphorylation. The putative autoinhibitory segment contains several serine and threonine residues, each followed directly by a proline residue. This motif may prevent autophosphorylation but permit transphosphorylation; two of these serine residues reside in a maturation promoting factor (MPF)/cdc-2 consensus motif. Thus, hormonal regulation of S6 kinase may involve the action of MPF/cdc-2 or protein kinases with related substrate specificity.     

Expression of 11beta-hydroxylase in rat Leydig cells.

11Beta-hydroxy (11beta-OH) derivatives of certain steroids function as inhibitors of 11beta-hydroxysteroid dehydrogenase isoform 1 (11betaHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11beta-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11beta-hydroxylation of steroids and thus producing 11beta-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11beta-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11beta-hydroxylated steroids inhibit Leydig cell 11betaHSD1 activities. 11beta-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11beta-hydroxylase protein in Leydig cells. Moreover, 11beta-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the K(m) and maximum velocity values were 7.28 +/- 0. 92 microM and 1.13 +/- 0.04 micromol/10(6) cell x h, respectively. When assayed in Leydig cells, several 11beta-hydroxylated steroids were efficient inhibitors of 11betaHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11beta-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11betaHSD1.     

3beta-hydroxysteroid dehydrogenase autoantibodies are rare in premature ovarian failure

Premature ovarian failure (POF) is a disorder of heterogeneous etiology, and autoimmunity has been suspected as one cause of POF. The steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD), has been characterized as a potential autoantigen in POF as well as in insulin-dependent diabetes mellitus (type 1 diabetes). Here we studied the presence of steroid cell antibodies (SCA), autoantibodies to 3betaHSD and to two other known autoantigens in ovarian failure, steroidogenic enzymes 17alpha-hydroxylase (P450c17), and side-chain cleavage enzyme (P450scc) in POF patients and patient groups with autoimmune polyendocrinopathy syndromes type 1 and 2 (APS1 and -2), isolated Addison's disease, type 1 diabetes, and healthy controls. The SCA were found in 2 of 48 POF, 11 of 15 APS1, and 1 of 9 APS2, and autoantibodies to in vitro translated 3betaHSD protein were detected in 1 POF serum associated with Addison's disease and 3 APS1 sera. All 3betaHSD precipitating sera were also positive for SCA. However, no SCA or 3betaHSD autoantibodies were found in 38 Addison's disease, 28 type 1 diabetes, and 71 healthy control sera. In analysis of autoantibodies to P450c17 and P450scc, antibodies to these enzymes were not found in POF sera, but were found in 10 and 12 APS1 patient sera, respectively, and 1 APS2 patient serum contained anti-P450c17 antibodies. Our results show that autoantibodies to 3betaHSD in POF patients are rare and are also found in patients with APS1.     

Complementary deoxyribonucleic acid cloning and enzymatic characterization of a novel 17beta/3alpha-hydroxysteroid/retinoid short chain dehydrogenase/reductase.

17Beta-hydroxysteroid dehydrogenases (17betaHSDs) convert androgens and estrogens between their active and inactive forms, whereas retinol dehydrogenases catalyze the conversion between retinol and retinal. Retinol dehydrogenases function in the visual cycle, in the generation of the hormone retinoic acid, and some also act on androgens. Here we report cloning and expression of a complementary DNA that encodes a new mouse liver microsomal member of the short chain dehydrogenase/reductase (SDR) superfamily and its enzymatic characterization, i.e. 17betaHSD9. Although 17betaHSD9 shares 88% amino acid identity with rat 17betaHSD6, its closest homolog, the two differ in substrate specificity. In contrast to other 17betaHSD, 17betaHSD9 has nearly equivalent activities as a 17betaHSD (with estradiol approximately = adiol) and as a 3alphaHSD (with adiol approximately = androsterone). It also recognizes retinol as substrate and represents in part the NAD+-dependent liver microsomal dehydrogenase that uses unbound retinol, but not retinol complexed with cellular retinol-binding protein. Thus, this enzyme has catalytic properties that overlap with two subgroups of SDR, 17betaHSD and retinol dehydrogenases. Inactivation of estrogen and a variety of androgens seems to be its most probable function. Because of its apparent inability to access retinol bound with cellular retinol-binding protein, a function in the pathway of retinoic acid biosynthesis seems less obvious. These data provide additional insight into the enzymology of estrogen, androgen, and retinoid metabolism and illustrate how closely related members of the SDR superfamily can have strikingly different substrate specificities.     

New inhibitors of 17beta-hydroxysteroid dehydrogenase type 1

The estradiol-synthesizing enzyme 17beta-hydroxysteroid dehydrogenase type 1 (17betaHSD1) is mainly responsible for the conversion of estrone (E1) to the potent estrogen estradiol (E2). It is a key player to control tissue levels of E2 and is therefore an attractive target in estradiol-dependent diseases like breast cancer or endometriosis. We selected a unique non-steroidal pyrimidinone core to start a lead optimization program. We optimized this core by modulation of R1-R6. Its binding mode at the substrate-binding site of 17betaHSD1 is complex and difficult to predict. Nevertheless, some basic structure-activity relationships could be identified. In vitro, the most active pyrimidinone derivative showed effective inhibition of recombinant human 17betaHSD1 at nanomolar concentrations. In intact cells overexpressing the human enzyme, IC50 values in the lower micromolar range were determined. Furthermore, the pyrimidinone proved its use in vivo by significantly reducing 17betaHSD1-dependent tumor growth in a new nude mouse model.     

Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.

The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases.     

Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte.

Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.     

A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 is highly homologous to vertebrate cyclophilin B.

A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1. The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8. Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM. The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs. By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated. It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal. The Orpinomyces PC-2 CyP is a typical type B CyP. The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins. Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.     

The complete amino acid sequence of the follitropin beta-subunit of the bullfrog, Rana catesbeiana.

The amino acid sequence of the bullfrog, Rana catesbeiana, follitropin beta-subunit has been determined by sequencing the intact protein (residues 1-39) and peptides originated by lysyl endopeptidase and pepsin. Twelve cysteine residues and two sugar chain binding sites at Asn-5 and Asn-22 are positional identities with bullfrog and mammalian beta-subunits. The bullfrog FSH beta-subunit is composed of 107 amino acid residues with a molecular mass of 11,782 Da, including the six cystine bridges and excepting the sugar chain. The bullfrog FSH beta-subunit has approximately 60% sequence identity with that of mammals and 40% with the fish gonadotropin beta-subunit. Conserved sequences among mammals (residue numbers 33-55 and 66-71) extensively differed from those of the bullfrog.     

Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain.

The brain is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Although considerable information is now available regarding the steroidogenic capacity of the brain, little is known regarding the steroidogenic pathway and relative contributions of astrocytes, oligodendrocytes, and neurons to neurosteroidogenesis. In the present study, we investigated differential gene expression of the key steroidogenic enzymes using RT-PCR and quantitatively evaluated the production of neurosteroids by highly purified astrocytes, oligodendrocytes, and neurons from the cerebral cortex of neonatal rat brains using specific and sensitive RIAs. Astrocytes appear to be the most active steroidogenic cells in the brain. These cells express cytochrome P450 side-chain cleavage (P450scc), 17alpha-hydroxylase/C17-20-lyase (P450c17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17beta-hydroxysteroid dehydrogenase (17betaHSD), and cytochrome P450 aromatase (P450arom) and produce pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), estradiol, and estrone. Oligodendrocytes express only P450scc and 3betaHSD and produce P5, P4, and A4. These cells do not express P450c17, 17betaHSD, or P450arom or produce DHEA, T, or estrogen. Neurons express P450scc, P450c17, 3betaHSD, and P450arom and produce P5, DHEA, A4, and estrogen, but do not express 17betaHSD or produce T. By comparing the ability of each cell type in the production of neurosteroids, astrocytes are the major producer of P4, DHEA, and androgens, whereas oligodendrocytes are predominantly the producer of P5 and neurons of estrogens. These findings serve to define the neurosteroidogenic pathway, with special emphasis on the dominant role of astrocytes and their interaction with oligodendrocytes and neurons in the genesis of DHEA and active sex steroids. Thus, we propose that neurosteroidogenesis is accomplished by a tripartite contribution of the three cell types in the brain.     

Human Stomach Class IV Alcohol Dehydrogenase: Molecular Genetic Analysis

A partial human stomach alcohol dehydrogenase (ADH) encoding cDNA has been isolated, cloned, and sequenced, which contains 222 nucleotides encoding amino acid residues 227–299 of the ADH subunit. The amino acid sequence deduced from this cDNA was highly homologous with the rat stomach class IV ADH sequence recently reported (81.1% sequence identity). Homology with other human ADH classes was also observed: class I, 58.1% sequence identity; class II, 39.2% sequence identity; class III, 55.4% sequence identity; and class V, 50.0% sequence identity. These results support a proposal that the isolated cDNA encodes a partial sequence for human stomach class IV ADH. This sequence retains val294 for all other human ADH classes reported, as compared with an ala294 at this position reported for rat class IV ADH. This ala residue may contribute to the very high K_m values with ethanol for the latter enzyme. In addition, three substitutions are reported for key residues in the coenzyme binding site: 251, gln/ser; 260, gly/asn; and 261, gly/asn, which may contribute to the weak coenzyme binding properties reported for human class IV ADH.