Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.3 (HSD)
3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzyme, rat ovarian 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD), plays a central role in luteolysis and parturition. It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20 alpha HSD. To further investigate whether phosphorylation and/or glycosylation affect the activity of 20 alpha HSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme. The recombinant (rec) 20 alpha HSD expressed as glutathione-S-transferase-20 alpha HSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatographies. Both rec preparations of 20 alpha HSD demonstrated a single polypeptide chain of 37 kDa with similar K(m) values for 20 alpha-hydroxyprogesterone and NADP, although the corresponding maximum velocity values were slightly lower for the rec 20 alpha HSD expressed in the insect cells. The rec 20 alpha-HSD showed preference for progesterone/20 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was only 30% as effective. The enzyme also used various substrates specific for aldo-keto reductases, although with much less efficiency. The rec enzyme preparations showed an absolute requirement for NADP(H). In vitro phosphorylation of rec bacterial enzyme with either protein kinase A or protein kinase C had no demonstrable effect on its activity. Finally, no differences in enzyme activity were noted between glycosylated (expressed in insect cells) and nonglycosylated (expressed in bacteria) forms of the enzyme. In conclusion, these studies demonstrate that rat luteal 20 alpha HSD can be prepared in large amounts from either bacterial or insect expression systems in a catalytically active form. Indirect evidence also suggests that the catalytic activity of 20 alpha HSD may be independent of phosphorylation and glycosylation states of the enzyme protein, i.e. posttranslational modification of 20 alpha HSD may not be required for the maximal expression of enzyme activity.
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PMID:Expression, purification and characterization of the rat luteal 20 alpha-hydroxysteroid dehydrogenase. 897 2

Corticosteroids (glucocorticoids and mineralocorticoids) have multiple actions in the brain which are mediated via specific intracellular receptors. Recently, a novel and important control of glucocorticoid action has been identified in peripheral tissues; prereceptor metabolism by 11beta-hydroxysteroid dehydrogenase (11beta-HSD). This enzyme catalyses the conversion of the active glucocorticoids corticosterone and cortisol to inert 11 keto-products (11-dehydrocorticosterone, cortisone), thus regulating access of glucocorticoids to receptors. Two distinct isozymes occur. 11beta-HSD-1 is a widespread, NADP(H)-dependent enzyme, which shows bidirectional activity in tissue homogenates and microsomal preparations, but may predominantly function as an 11beta-reductase (regenerating active glucocorticoids) in intact cells. 11beta-HSD-2 is a much higher affinity, NAD-dependent, exclusive 11beta-dehydrogenase (glucocorticoid inactivating enzyme), which, when colocalized with otherwise nonselective mineralocorticoid receptors (MR), ensures selective access for aldosterone in vivo. Accumulating evidence indicates widespread expression of 11beta-HSD-1 in the brain. The highest levels are found in cerebellum, hippocampus, cortex, and pituitary, but detectable activity is also present in the hypothalamus (including the paraventricular nucleus) and other regions of neuroendocrine interest. 11beta-HSD-1 protein has been detected on Western blots of brain and immunostaining is widespread, localized predominantly in neurons and their processes. The mRNA encoding 11beta-HSD-1 is also widely expressed in the brain, its distribution broadly paralleling enzyme bioactivity and immunostaining. 11beta-HSD-1 expression is regulated during late prenatal and postnatal ontogeny and by glucocorticoids and stress, prompting suggestions that this isoform may play a role in protecting the brain from the deleterious consequences of glucocorticoid excess. However, in primary cultures of hippocampal neurons, 11beta-HSD-1 functions as a predominant 11beta-reductase, reactivating inert corticoids and thus potentiating neurotoxicity. The functions of 11beta-HSD-1 in the CNS are not defined, but may relate to mood, neuronal survival, and glucocorticoid feedback. The identification of aldosterone-selective actions in the brain (upon blood pressure and salt appetite) predict the presence of 11beta-HSD-2. This isozyme has very limited expression in the adult brain, probably confined to the subregions of the brain stem and the subcommissural organ, where some aldosterone-selective actions may be mediated. However, the midgestation fetal brain highly expresses 11beta-HSD-2, which might modulate glucocorticoid effects on CNS development. Studies with licorice-derived enzyme inhibitors indicate functional effects for 11beta-HSD in the adult brain, notably in the periventricular hypothalamus and limbic system. Thus, 11beta-HSD represents a novel and potentially important level of control of glucocorticoid action in the CNS. Enzyme modulation by pharmacological or other agents may provide a useful means to target increased or attenuated glucocorticoid action to specific sites in the brain.
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PMID:11beta-Hydroxysteroid dehydrogenase in the brain: a novel regulator of glucocorticoid action? 900 Apr 59

Mammalian 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSDs) inactivate circulating steroid hormones, and in target tissues regulate the occupancy of steroid hormone receptors. Molecular cloning indicates that 3 alpha-HSDs are members of the aldo-keto reductase (AKR) superfamily and display high sequence identity (> 60%). Of these, the most extensively characterized is rat liver 3 alpha-HSD. X-ray crystal structures of the apoenzyme and the E.NADP+ complex have been determined and serve as structural templates for other 3 alpha-HSDs. These structures reveal that rat liver 3 alpha-HSD adopts an (alpha/beta)8-barrel protein fold. NAD(P)(H) lies perpendicular to the barrel axis in an extended conformation, with the nicotinamide ring at the core of the barrel, and the adenine ring at the periphery of the structure. The nicotinamide ring is stabilized by interaction with Y216, S166, D167, and Q190, so that the A-face points into the vacant active site. The 4-pro-(R) hydrogen transferred in the oxidoreduction of steroids is in close proximity to a catalytic tetrad that consists of D50, Y55, K84, and H117. A water molecule is within hydrogen bond distance of H117 and Y55, and its position may mimic the position of the carbonyl of a 3-ketosteroid substrate. The catalytic tetrad is conserved in members of the AKR superfamily and resides at the base of an apolar cleft implicated in binding steroid hormone. The apolar cleft consists of a side of apolar residues (L54, W86, F128, and F129), and opposing this side is a flexible loop that contains W227. These constraints suggest that the alpha-face of the steroid would orient itself along that side of the cleft containing W86. Site-directed mutagenesis of the catalytic tetrad indicates that Y55 and K84 are essential for catalysis. Y55S and Y55F mutants are catalytically inactive, but still form binary (E.NADPH) and ternary (E.NADH.Testosterone) complexes; by contrast K84R and K84M mutants are catalytically inactive, but do not bind steroid hormone. The reliance on a Tyr/Lys pair is reminiscent of catalytic mechanisms proposed for other AKR members as well as for HSDs that belong to the short-chain dehydrogenase/reductase (SDR) family, in which Tyr is the general acid, with its pKa being lowered by Lys. Superimposition of the nicotinamide rings in the structures of 3 alpha-HSD (an AKR) and 3 alpha, 20 beta-HSD (an SDR) show that the Tyr/Lys pairs are positionally conserved, suggesting convergent evolution across protein families to a common mechanism for HSD catalysis. W86Y and W227Y mutants bind testosterone to the E.NADH complex, with effective increases in Kd of 8- and 20-fold. These data provide the first evidence that the side of the apolar cleft containing W86 and the opposing flexible loop containing W227 are parts of the steroid-binding site. Detailed mutagenesis studies of the apolar cleft and elucidation of a ternary complex structure will ultimately provide details of the determinants that govern steroid hormone recognition. These determinants could provide a rational basis for structure-based inhibitor design.
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PMID:Structure and function of 3 alpha-hydroxysteroid dehydrogenase. 902 23

Whereas aldosterone is normally a much stronger mineralocorticoid than cortisol in vivo, mineralocorticoid receptors have identical in vitro affinities for these hormones. The in vivo specificity of the receptors is, at least in part, the result of activity of 11-HSD, an enzyme located in most mineralocorticoid target tissues that converts cortisol to cortisone. Cortisone is not a ligand for the receptor, whereas aldosterone is not a substrate of the enzyme. The syndrome of AME is a rare form of juvenile hypertension in which 11-HSD is defective. This deficiency allows mineralocorticoid receptors to be occupied by cortisol, leading to hypertension, because plasma concentrations of cortisol are much higher than those of aldosterone. Licorice, which contains 11-HSD inhibitors, causes a similar syndrome. There are two known isozymes of 11-HSD. The liver or type I isozyme is expressed at high levels in the liver, has a relatively low affinity for steroids (micromolar Km), catalyzes both dehydrogenation and the reverse reductase reaction, and utilizes NADP+ or NADPH as cofactors. The kidney or type 2 isozyme is expressed at high levels in the kidney and placenta, has a high affinity (nanomolar Km) for steroids, catalyzes only dehydrogenation, and utilizes NAD+ as a cofactor. Mutations in the HSD11B2 (HSD11K) gene encoding the kidney isozyme of 11-HSD have been detected in all kindreds with AME studied thus far. This gene represents a candidate locus for the common, "essential" form of hypertension.
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PMID:11 beta-Hydroxysteroid dehydrogenase and the syndrome of apparent mineralocorticoid excess. 903 89

We have previously reported that 5 alpha and 5 beta pathways of steroid metabolism are controlled in vivo by dietary Na+ and glycyrrhetinic acid, see Gorsline et al. 1988; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11 beta-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11 beta-HSD2 (kidney). The activity of 11 beta-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11 beta-HSD2 is particularly affected. In all three conditions, excretion of the less common 5 alpha metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5 alpha and 5 beta) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP+ as co-factor (11 beta-HSD1), and sheep kidney microsomes with NAD+ as co-factor (11 beta-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11 beta-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5 alpha and 5 beta derivatives. 11 beta-HSD1 is inhibited by both 5 alpha and certain 5 beta derivatives. 11 beta-HSD-2 was selectively inhibited only by 5 alpha derivatives: 5 beta derivatives were without inhibitory activity toward this isoform of 11 beta-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11 beta-HSD1 and 11 beta-HSD2.
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PMID:Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5 alpha-reduced (but not 5 beta) derivatives of adrenocorticosteroids. 905 82

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the enzyme that catalyzes the conversion of biologically active glucocorticoids to their inactive metabolites, was shown to be located exclusively in Leydig cells of the rat testis, and its appearance was associated with the developmental rise in testosterone. Thus, 11 beta-HSD was suggested to play an important role in maintaining steroidogenesis by inactivating excess cortisol that inhibits testosterone production. Whether equivalent protection from glucocorticoids excess is necessary for spermatogenesis is not known, and we have, accordingly, investigated the 11 beta-HSD activity in ejaculated human semen. Both 11 beta-dehydrogenase (11 beta-DH) and 11 beta-oxoreductase (11-OR) activities of 11 beta-HSD were measurable in semen, although seminal plasma was devoid of 11 beta-HSD activity. Azoospermic specimens were associated with low 11 beta-dehydrogenase activity, indicating the presence of enzyme activity in cells other than spermatozoa. Pure spermatozoa separated on percoll gradient could oxidize corticosterone in the presence of NAD or NADP. Significantly higher 11 beta-DH activity is associated with semen specimens with low sperm count (p < .05) and higher level of morphologically abnormal spermatozoa (p < .05). The presence of 11 beta-HSD in human semen and its association with sperm characteristics thus suggests functional role for glucocorticoid exclusion in the sperm maturation process.
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PMID:Presence of 11 beta-hydroxysteroid dehydrogenase in human semen: evidence of correlation with semen characteristics. 907 40

11 beta-HSD catalyses the interconversion of active and inactive corticosteroids and exists as two isoforms with less than 30% amino acid homology. The bi-directional NADP-dependent type 1 enzyme appears to function as a tissue-specific glucocorticoid provider. The uni-directional NAD-dependent type 2 enzyme functions as a tissue-specific glucocorticoid protector. The syndrome of AME is caused by mutations in the gene of 11 beta-HSD2. Placental 11 beta-HSD2 is a barrier to growth-retarding maternal glucocorticoids and may play a key role in prenatal programming of hypertension.
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PMID:11 beta-Hydroxysteroid dehydrogenases: tissue-specific dictators of glucocorticoid action. 907 55

11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the interconversion of cortisol to hormonally inactive cortisone (corticosterone (B) to 11-dehydrocorticosterone (A) in rodents), and as such is established as a pre-receptor signalling pathway for corticosteroid hormone action. To further evaluate the role of this enzyme in adult and fetal life we have characterized two isoforms of 11beta-HSD in mouse tissues. Mouse 'liver' or type 1 11beta-HSD is a bi-directional dehydrogenase/oxo-reductase (K(m) for B 1.9 microM, K(m) for A 0.73 microM). Oxo-reductase activity utilized only NADPH as a co-factor, whilst dehydrogenase activity increased with both NAD or NADP. Mouse 'kidney' or 11beta-HS3D2 activity was NAD-dependent with a K(m) for B of 0.11 microM. Dexamethasone was not a substrate. Using an in-house mouse 11beta-HSD2 cDNA and NAD-dependent activity studies, 11 beta-HSD2 was expressed in epithelial cells of colon, renal collecting ducts, ovary, and adrenal, but was absent in liver, spleen, testis and heart. With the exception of gonadal tissues, activity and mRNA levels were consistently higher in adult male versus female tissues. In fetal kidney and colon there was absent/low levels of 11beta-HSD2 expression from fetal day 15 to term (day 19/20). Placental 11beta-HSD2 mRNA and activity were highest on fetal day 13/14 and fell progressively to undetectable levels by term. Two isoforms of 11beta-HSD are present in mouse tissues in accordance with other mammalian species. The sexual-dimorphic expression 11 beta-HSD2 in kidney and colon may reflect male-female differences in sodium homeostasis, and the absent expression of 11 beta-HSD2 in late gestation may facilitate glucocorticoid-dependent maturation of mouse fetal tissues.
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PMID:Ontogeny and sexual dimorphic expression of mouse type 2 11beta-hydroxysteroid dehydrogenase. 909 7

Endogenous glucocorticoids are converted to their biologically inert 11-dehydroderivatives by isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). The low-K(m), NAD(+)-dependent renal isoform (Type 2) identified in the distal nephron protects mineralocorticoid receptors from activation by endogenous glucocorticoids. The function of high-K(m), NADP(+)-dependent renal isoform (Type 1) is less well understood. Since glucocorticoids may modulate sodium transport in renal proximal tubules (PT), we hypothesized that Type 1 activity in this segment may be regulated by dietary Na(+)-11 beta-HSD activity was assessed in homogenates of canine PT by the conversion of cortisol to cortisone in the presence of NADP+ 200 microM. A high-Na+ diet for 4 days increased the Vmax 4-fold, with no change in the Type 1 K(m) (40 mEq/day Na+ diet: K(m) 0.959 microM, Vmax 3.40 pmoles/min/mg protein versus 150 mEq/day Na+ diet: K(m) 0.962 microM, Vmax 14.8 pmoles/min/mg protein). Type 1 mRNA also rose in the salt repleted animals. The high-Na+ diet produced no detectable change in the Type 2 isoform enzyme kinetics and mRNA level. No reverse oxo-reductase activity was noted with either renal isoform. Thus, renal Type 1 11 beta-HSD can be regulated by dietary Na+ independent of changes in the renal Type 2 isoform.
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PMID:Influence of dietary sodium on the renal isoforms of 11 beta-hydroxysteroid dehydrogenase. 911 24

Dexamethasone is used in the clinic to test the sensitivity of the hypothalamic-pituitary-adrenal axis to negative feedback. It has also been proposed that metabolism of dexamethasone might differentiate between the activities of the two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD1 and 11 beta HSD2). We have developed a gas chromatographic mass spectrometric assay for dexamethasone and 11-dehydrodexamethasone and have confirmed in vitro that dexamethasone is a substrate for 11 beta-HSD2 but not 11 beta-HSD1 (conversion to 11-dehydrodexamethasone 0.6 +/- 0.3% in homogenates of rat liver with NADP+ for 11 beta-HSD1, and 29.4 +/- 10.3% and 40.0 +/- 2.0% in homogenates of rat and human kidney respectively with NAD+ for 11 beta-HSD2). However, we have also made the novel observation that 11-dehydrodexamethasone is a substrate for both isozymes (conversion to dexamethasone 65.0 +/- 20.4% for 11 beta HSD1 and 53.5 +/- 20.8% and 69.0 +/- 4.5% for 11 beta HSD2, rat and human respectively). In healthy humans, the concentrations of 11-dehydrodexamethasone in plasma after an intravenous bolus of dexamethasone were less than 10% of those of dexamethasone, and 11-dehydrodexamethasone was detected (at 0.8-65.0 nM) in plasma from only 11 of 20 subjects at 0900 h on the morning after oral dexamethasone (0.1-1 mg taken at 2400 h). Concentrations of 11-dehydrodexamethasone did not correlate with the degree of suppression of plasma cortisol. Thus dexamethasone is not useful in differentiating the activities of the isozymes of 11 beta-HSD in vivo and variations in 11 beta-HSD activity do not explain the interindividual variability in suppression of plasma cortisol by low doses of dexamethasone.
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PMID:Dexamethasone and 11-dehydrodexamethasone as tools to investigate the isozymes of 11 beta-hydroxysteroid dehydrogenase in vitro and in vivo. 913 68


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