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PubMed1981

Affinity labeling of human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

Two pyridine nucleotide linked oxidoreductase activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, which were copurified from human placental cytosol as a homogeneous enzyme preparation, may represent dual activity by one enzyme. The affinity labeling nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, which binds at the cofactor site as a competitive inhibitor of NADH (ki = 1.7 mM), simultaneously and identically inactivated both the 17 beta and 20 alpha activities in a time-dependent and irreversible manner following pseudo-first-order kinetics. NADH and NAD+ markedly protected both activities from inactivation, and the substrate steroids, estrone, estradiol, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one, conferred similar protection, though less than cofactor, against simultaneous loss of both activities. Stoichiometric studies indicated that 2 mol of affinity labeling nucleotide were bound per mol of completely inactivated enzyme dimer. The coincident and identical loss of both activities under all experimental conditions is further evidence that 17 beta-estradiol dehydrogenase and 20 alpha-hydrosteroid dehydrogenase in human placental cytosol represent bifunctional, stereospecific, oxidoreductase activity at one active site on a single protein.

17-Hydroxysteroid Dehydrogenases↗
PubMed1978

Comparison of the 3alpha-and 20beta-hydroxysteroid dehydrogenase activities of the cortisone reductase of Streptomyces hydrogenans.

The 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSD) activities of cortisone reductase in Streptomyces hydrogenans have been examined to determine whether both activities are due to one enzyme. This question was raised when changes in the commercial preparations of the enzyme reduced the 3alpha-HSD activity to 5% of its original level while retaining full 20beta-HSD activity. In our experiments, the enzyme was purified to crystallinity and partially characterized. The 3alpha- and 20beta-HSD activities were both coinduced and copurified. The 3alpha- and 20beta-HSD activities were compared using the crystalline enzyme for studies of substrate competition, thermal inactivation at 52 degrees C, loss of activity with three haloacetoxysteroids, and the effects of Me2SO and temperature on the reaction rate. These studies support the conclusion that the 3alpha- and 20beta-HSD activities are due to the same enzyme molecule. In addition, it appears that the binding sites for the two activities do not act independently.

20-Hydroxysteroid Dehydrogenases↗
PubMed2001

Crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP(+) and ursodeoxycholate.

The crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP(+) and 3alpha,7beta-dihydroxy-5beta-cholanic acid (ursodeoxycholate) at 3.0 A resolution is presented. Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily. AKR1C2 is implicated in the prostatic production of the potent androgen 5alpha-dihydrotestosterone and the hepatic transport of bile acids. It also catalyzes the formation of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria. Like other members of the superfamily, AKR1C2 folds into an alpha/beta-barrel and binds NADP(+) in an extended conformation. The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site. More interestingly, the orientation of ursodeoxycholate is essentially "backwards" and "upside-down" from that observed for testosterone in the related rat 3alpha-HSD.NADP(+).testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate. The orientation of ursodeoxycholate is thus similar to that expected of a 17beta-HSD substrate. The ternary structure explains the ability of AKR1C2 to catalyze 3alpha-, 17beta-, and 20alpha-HSD reactions. Comparison of the steroid binding pocket of AKR1C2 with that of rat 3alpha-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3alpha-HSD isoforms but not in the rat isoform.

Allosteric Regulation↗
PubMed2002

11 Beta-hydroxysteroid dehydrogenase type 1 from human liver: dimerization and enzyme cooperativity support its postulated role as glucocorticoid reductase.

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme that catalyzes the reversible interconversion of receptor-active 11-hydroxy glucocorticoids (cortisol) to their receptor-inactive 11-oxo metabolites (cortisone). However, the physiological role of 11beta-HSD 1 as prereceptor control device in regulating access of glucocorticoid hormones to the glucocorticoid receptor remains obscure in light of its low substrate affinities, which is in contrast to low glucocorticoid plasma levels and low Kd values of the receptors to cortisol. To solve this enigma, we performed detailed kinetic analyses with a homogeneously purified 11beta-HSD 1 from human liver. The membrane-bound enzyme was successfully obtained in an active state by a purification procedure that took advantage of a gentle solubilization method as well as providing a favorable detergent surrounding during the various chromatographic steps. The identity of purified 11beta-HSD 1 was proven by determination of enzymatic activity, N-terminal amino acid sequencing, and immunoblot analysis. By gel-permeation chromatography we could demonstrate that 11beta-HSD 1 is active as a dimeric enzyme. The cDNA for the enzyme was cloned from a human liver cDNA library and shown to be homologous to that previously characterized in human testis. Interestingly, 11beta-HSD 1 exhibits Michaelis-Menten kinetics with cortisol and corticosterone (11beta-dehydrogenation activity) but cooperative kinetics with cortisone and dehydrocorticosterone (11-oxoreducing activity). Accordingly, this enzyme dynamically adapts to low (nanomolar) as well as to high (micromolar) substrate concentrations, thereby providing the fine-tuning required as a consequence of great variations in circadian plasma glucocorticoid levels.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2003

Functional characterization of the human 11 beta-hydroxysteroid dehydrogenase 1B (11 beta-HSD 1B) variant.

11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD 1) catalyzes the interconversion of inactive into active glucocorticoids and has been shown to play a key role in metabolic disorders such as obesity and diabetes. 11 beta-HSD 1 belongs to the short chain dehydrogenases/reductases (SDR) and shares all common structural motifs typically for this protein superfamily. Unlike common SDRs, 11 beta-HSD 1 is N-terminally extended by a hydrophobic domain that anchors this enzyme in the endoplasmic reticulum (ER) membrane. Interestingly, the occurrence of 11 beta-HSD 1 transcripts lacking the N-terminal hydrophobic domain has repeatedly been reported in a variety of tissues, and the corresponding protein has been named 11 beta-HSD 1B. So far, no activity of 11 beta-HSD 1B has been observed, such that a physiological role could not be ascribed. In the present investigation, we showed for the first time that the truncated human 11 beta-HSD 1B form, expressed in the yeast Pichia pastoris, may indeed be active. However, this activity was prevented by the fact that 11 beta-HSD 1B is still kept attached to the ER membrane. Via computer assisted simulation and modeling, we identified a putative domain within the 11 beta-HSD 1 structure that could be responsible for this additional membrane attachment. By performing site-directed mutagenesis, heterologous expression, immunoblot analysis, and activity assays, we verified that this hydrophobic domain could indeed interact with the ER membrane and that some of the introduced mutations (V149R, V149E) led to a release of 11 beta-HSD 1B from membrane attachment without affecting its enzymatic activity. However, the activity of 11 beta-HSD 1B proved to be very unstable and was lost within hours after solubilization and release from the ER membrane. Importantly, 11 beta-HSD 1 constructs lacking the first 15 N-terminal amino acids and bearing additional amino acid substitutions (t15-V149R, t15-V149E) were then found to be soluble and to be stable in terms of enzyme activity. Combined, despite its occurrence in mammalian tissues, 11 beta-HSD 1B has obviously no physiological role since it is either inactive while being attached to the ER or it is rapidly losing activity once being released from intracellular membranes. Our findings with the t15-V149R and t15-V149E constructs are promising to further understand the complex mechanical and structural properties of 11 beta-HSD 1.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1998

Mutagenesis of 3 alpha-hydroxysteroid dehydrogenase reveals a "push-pull" mechanism for proton transfer in aldo-keto reductases.

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, E.C. 1.1.1.213, AKR1C9) is a member of the aldo-keto reductase (AKR) superfamily which inactivates circulating steroid hormones. We have proposed a catalytic mechanism in which Tyr 55 acts as a general acid with its pK value being lowered by a hydrogen bond with Lys 84 which is salt-linked to Asp 50. To test this mechanism, residues at the active site were mutated and the mutant enzymes (Y55F, Y55S, K84M, K84R, D50N, D50E, and H117A) were purified to homogeneity from an Escherichia coli expression system. Spectrophotometric assays showed that mutations of Tyr 55 and Lys 84 gave enzymes that were apparently inactive for steroid oxidation and reduction. All mutants appeared inactive for steroid reduction. The catalytic efficiencies for steroid oxidation were reduced 4-10-fold for the Asp 50 mutants and 300-fold for the H117A mutant. Fluorescence titration with NADPH demonstrated that each mutant bound cofactor unimpeded. Equilibrium dialysis indicated that the competitive inhibitor testosterone formed E.NADH.testosterone complexes only with the Y55F, Y55S, and D50N mutants with Kd values 10-fold greater than those for wild-type. Therefore the loss of steroid oxidoreductase activity observed for the Tyr 55 mutants cannot be attributed simply to an inability to bind steroid. Using a highly sensitive radiometric assay in which the conversion of [14C]-5 alpha-dihydrotestosterone (DHT) to [14C]-3 alpha-androstanediol (3 alpha-Diol) was measured, the rate enhancement (kcat/knoncat) for the reaction was estimated to be 2.6 x 10(9). Using this assay, all mutants formed steroid product with decreases in an overall rate enhancement of 10(1)-10(4). It was found that Tyr 55 made the single largest contribution to rate enhancement. This is the first instance where point mutations in the conserved catalytic tetrad of an AKR yielded enzymes which were still catalytically active. This enabled the construction of pH vs kcat profiles for the reduction of [14C]-5 alpha-DHT catalyzed by the tetrad mutants. These profiles revealed that the titratable group assigned to the general acid (pK = 6.50 +/- 0.42) was eliminated in the Y55F and H117A mutants. The pH-independent value of kcat was decreased in the H117A and Y55F mutants, by 2 and 4 log units, respectively. pH vs kcat(app) profiles for the oxidation of [3H]-3 alpha-Diol showed that the same titratable group (pK = 7.50 +/- 0.30) was eliminated in both the Y55F and K84M mutants but was retained in the H117A mutant. Since only the Y55F mutant eliminated the titratable group in both the reduction and oxidation directions it is assigned as the catalytic general acid/base. The differential effects of His 117 and Lys 84 on the ionization of Tyr 55 are explained by a "push-pull" mechanism in which His 117 facilitates proton donation and Lys 84 facilitates proton removal by Tyr 55.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Engineering steroid 5 beta-reductase activity into rat liver 3 alpha-hydroxysteroid dehydrogenase.

Delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) precedes 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) in steroid hormone metabolism. Both enzymes are members of the aldo-keto reductase (AKR) superfamily and possess catalytic tetrads differing by a single amino acid. In 3 alpha-HSD, the tetrad consists of Tyr55, Lys84, Asp50, and His117, but a glutamic acid replaces His117 in 5 beta-reductase. By introducing the H117E point mutation into 3 alpha-HSD, we engineered 5 beta-reductase activity into the dehydrogenase. Homogeneous H117E 3 alpha-HSD reduced the double bond in testosterone to form 5 beta-dihydrotestosterone with kcat = 0.25 min-1 and Km = 19.0 microM and reduced the double bond in progesterone to generate 5 beta-dihydroprogesterone with kcat = 0.97 min-1 and Km = 33.0 microM. These kinetic parameters were similar to those reported for homogeneous rat liver 5 beta-reductase [Okuda, A., and Okuda, R. (1984) J. Biol. Chem. 259, 7519-7524]. The H117E mutant also reduced 5beta-dihydrosteroids to 5 beta, 3 alpha-tetrahydrosteroids with a 600-1000-fold decrease in kcat/Km versus wild-type 3 alpha-HSD. The ratio of 5 beta-reductase:3 alpha-HSD activity in the H117E mutant was approximately 1:1. Although the H117A mutant reduced Delta 4-3-ketosteroids, the 3 alpha-HSD activity predominated because the 5 beta-dihydrosteroids were rapidly converted to the 5 beta,3 alpha-tetrahydrosteroids. The pH-rate profiles for carbon-carbon double-bond and ketone reduction catalyzed by the H117E mutant were superimposable, suggesting a common titratable group (pKb = 6.3) for both reactions. In wild-type 3 alpha-HSD, the titratable group responsible for 3-ketosteroid reduction has a pKb = 6.9 and is assignable to Tyr55. The pH-rate profiles for 3-ketosteroid reduction by the H117A mutant were pH-independent. Our data indicate that Tyr55 functions as a general acid for both 3 alpha-HSD and 5 beta-reductase activities. We suggest that a protonated Glu117 increases the acidity of Tyr55 to promote acid-catalyzed enolization of the Delta 4-3-ketosteroid substrate. Further, the identity of amino acid 117 determines whether an AKR can function as a 5 beta-reductase by reorienting the substrate relative to the nicotinamide cofactor. This study provides functional evidence that utilization of modified catalytic residues on an identical protein scaffold is important for evolution of enzymatic activities within the same metabolic pathway.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Mutation of nicotinamide pocket residues in rat liver 3 alpha-hydroxysteroid dehydrogenase reveals different modes of cofactor binding.

Rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), an aldo-keto reductase, binds NADP(+) in an extended anti-conformation across an (alpha/beta)(8)-barrel. The orientation of the nicotinamide ring, which permits stereospecific transfer of the 4-pro-R hydride from NAD(P)H to substrate, is achieved by hydrogen bonds formed between the C3-carboxamide of the nicotinamide ring and Ser 166, Asn 167, and Gln 190 and by pi-stacking between this ring and Tyr 216. These residues were mutated to yield S166A, N167A, Q190A, and Y216S. In these mutants, K(d)(NADP(H)) increased by 2-11-fold but without a significant change in K(d)(NAD(H)). Steady-state kinetic parameters showed that K(m)(NADP)()+ increased 13-151-fold, and this was accompanied by comparable decreases in k(cat)/K(m)(NADP)()+. By contrast, K(m)(NAD)()+ increased 4-8-fold, but changes in k(cat)/K(m)(NAD)()+ were more dramatic and ranged from 23- to 930-fold. Corresponding changes in binding energies indicated that each residue contributed equally to the binding of NADP(H) in the ground and transition states. However, the same residues stabilized the binding of NAD(H) only in the transition state. These observations suggest that different modes of binding exist for NADP(H) and NAD(H). Importantly, these modes were revealed by mutating residues in the nicotinamide pocket indicating that direct interactions with the 2'-phosphate in the adenine mononucleotide is not the sole determinant of cofactor preference. The single mutations were unable to invert or racemize the stereochemistry of hydride transfer even though the nicotinamide pocket can accommodate both anti- and syn-conformers once the necessary hydrogen bonds are eliminated. When 4-pro-R-[(3)H]NADH was used to monitor incorporation into [(14)C]-5alpha-dihydrotestosterone, a decrease in the (3)H:(14)C ratio was observed in the mutants relative to wild-type enzyme reflecting a pronounced primary kinetic isotope effect. This observation coupled with the change in the binding energy for NAD(P)(H) in the transition state suggests that these mutants have altered the reaction trajectory for hydride transfer.

3-Hydroxysteroid Dehydrogenases↗
PubMed2002

Arylsulfonamidothiazoles as a new class of potential antidiabetic drugs. Discovery of potent and selective inhibitors of the 11beta-hydroxysteroid dehydrogenase type 1.

Novel antidiabetic arylsulfonamidothiazoles are presented that exert action through selective inhibition of the 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme, thereby attenuating hepatic gluconeogenesis. The diethylamide derivative 2a was shown to potently inhibit human 11beta-HSD1 (IC(50) = 52 nM), whereas the N-methylpiperazinamide analogue 2b only inhibited murine 11beta-HSD1 (IC(50) = 96 nM). Both compounds showed >200-fold selectivity over human and murine 11beta-HSD2. 2b was subsequently shown to reduce glucose levels in diabetic KKA(y) mice, substantiating the 11beta-HSD1 enzyme as a target for the treatment of type 2 diabetes.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed2001

3alpha-Hydroxysteroid dehydrogenase in animal and human tissues.

This review analyzes data on the biological role of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) in animal and human tissues and describes its main characteristics, mechanism of action, and regulation of activity. Based on published data, a scheme for the actions of androgen, progestin, and glucocorticoids involving the participation of 3alpha-HSD is proposed. According to this scheme, in the mechanism of steroid action 3alpha-HSD not only regulates the concentration of the main effector androgen, 5alpha-dihydrotestosterone, in target cells, but also switches androgen, progestin, and glucocorticosteroid genomic activity to non-genomic activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Multiple patterns of 11 beta-hydroxysteroid dehydrogenase catalytic activity along the mammalian nephron.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to be a protective enzyme of the mineralocorticoid receptor (MR). We have previously demonstrated (Bonvalet et al, J Clin Invest 86:832-837, 1990) that 11 beta-OHSD is colocalized with MR along the rabbit nephron. In the present study, we examined whether 11 beta-OHSD is similarly located along the nephron of other mammals. Various tubular segments were microdissected from the mouse, rat, and rabbit nephron, and incubated for two hours at 37 degrees C in the presence of 11 nM [3H]-corticosterone (B). Thereafter, the respective amounts of B and [3H]-11dehydrocorticosterone (A) in the incubation solution were measured by HPLC. In the rabbit, the mouse and the rat, about 520 pmol/10 mm of B were transformed into A in tubular segments possessing MR, that is, the distal parts of the nephron (distal and collecting tubule). Differences appeared in the aldosterone-insensitive proximal tubule; in both the initial and final parts of this segment, 11 beta-OHSD activity was low (26 pmol/10 mm) in the rabbit and the mouse, and relatively high in the rat (328 pmol/10 mm). In the cortical part of the loop of Henle, where the presence of MR is still under discussion, 11 beta-OHSD activity was low in the mouse (70 pmol/10 mm), high in the rat (533 pmol/10 mm) and intermediate in the rabbit (227 pmol/10 mm). The comparison of these results with previous data obtained with immunohistochemical methods suggests that the proximal and distal nephron might express different isoforms of 11 beta-OHSD.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

Developmental changes in rat renal 11 beta-hydroxysteroid dehydrogenase.

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) transforms endogenous glucocorticoids to their respective "biologically inert" 11-dehydro derivatives. A decrease in enzyme activity allows glucocorticoids to induce mineralocorticoid-like renal sodium retention. Since positive sodium balance is required for optimum growth in the newborn, we hypothesized that renal 11 beta-OHSD activity would be low in the postnatal period, a time of active growth. To test this, incubations with corticosterone were carried out using minces or homogenates prepared from kidneys of newborn, 8-day-old, and mature Sprague-Dawley rats. 11 beta-OHSD activity in renal minces, assessed by the percent of corticosterone (10(-8) M) transformed to 11-dehydrocorticosterone (compound A), was significantly lower in the newborn kidney (newborn 45.7 +/- 3.8%, 8 day 70.2 +/- 3.8%, and adult 73.4 +/- 3.1%, P < 0.001 1 vs. 8 day). Parallel studies were conducted using an antibody directed against liver 11 beta-OHSD counter stained with immunofluorescent labeled IgG. Kidneys from mature rats were brightly stained at S2 and S3 segments of proximal tubules. In contrast, staining was barely detectable in kidneys from the newborn and 8-day-old rats. When enzyme kinetics were examined in kidney homogenates (average protein concentration 2.5 mg/ml) in the presence of 200 microM NADP+, the apparent Km for corticosterone in the adult was 4.42 x 10(-6) M with a corresponding Vmax of 1.33 x 10(-9) mol/min/mg protein, while the apparent Km for corticosterone in the newborn was calculated to be 12.8 x 10(-8) M with a Vmax of 2.08 x 10(-11) mol/min/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Immunolocalization of NAD-dependent 11 beta-hydroxysteroid dehydrogenase in human kidney and colon.

The inactivation of physiological glucocorticoids by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) confers mineralocorticoid specificity to certain aldosterone target tissues. Both NADP, and NAD-dependent isoforms of 11 beta-HSD have been described. An NAD-dependent isoform of 11 beta-HSD (11 beta-HSD2) was recently cloned from human kidney. The present studies were designed to examine the cellular distribution of 11 beta-HSD2 in human kidney and colon, and to determine if the cellular distribution of 11 beta-HSD2 within the human kidney and colon is consistent with a role in conferring mineralocorticoid specificity. Using antibodies against a fusion protein containing a portion of the human 11 beta-HSD2, immunohistochemical staining of human kidney showed intense, specific staining of connecting tubules and cortical and medullary collecting tubules and less intense staining in the cortical thick ascending limb. No immunoreactivity was found in proximal tubules, glomeruli, or blood vessels. Within the collecting tubules staining was heterogeneous. The majority of cells showed intense cytoplasmic staining while alpha-intercalated cells displayed much less immunoreactivity. Within the colon, 11 beta-HSD2 immunoreactivity was found predominantly in surface epithelial cells but not in submucosal tissues. Thus, the distribution of the cloned NAD-dependent 11 beta-HSD2 parallels the distribution of mineralocorticoid receptors within the kidney and colon. These results support the view that the NAD-dependent isoform of 11 beta-HSD (11 beta-HSD2) provides mineralocorticoid specificity by inactivating glucocorticoids in an autocrine fashion.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

N-glycosylation is not essential for enzyme activity of 11beta-hydroxysteroid dehydrogenase type 2.

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the oxidation of cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. NAD-dependent 11beta-HSD is expressed at high levels in the distal nephron and contributes to mineralocorticoid specificity in that region. The present studies determined whether N-glycosylation is necessary for the activity of NAD-dependent 11beta-HSD (11beta-HSD2). First, cultured human colonic epithelial cells (T84 cells), which express native 11beta-HSD2 activity, were grown in medium with and without tunicamycin, an inhibitor of N-glycosylation. Tunicamycin had no effect on the enzyme activity. Next, the only putative N-glycosylation site (Asn394-Leu395-Ser396) of the cloned human kidney enzyme was eliminated by site-directed mutagenesis. Chinese hamster ovary (CHO) cells transfected with either the wild-type or the mutant cDNA construct showed no difference in the expressed enzyme activity, and Western blot analysis showed that the 11beta-HSD2 protein was the same size in cells expressing either the wild-type or the N394D mutant. Likewise, the molecular mass of the 11beta-HSD2 protein in T84 cells was not altered by treatment with peptide-N-glycosidase F or tunicamycin. We conclude that human 11beta-HSD2 is not a N-glycoprotein and N-glycosylation is not essential for the expression of enzyme activity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Human hypertension caused by mutations in the kidney isozyme of 11 beta-hydroxysteroid dehydrogenase.

The syndrome of apparent mineralocorticoid excess (AME) is an inherited form of human hypertension thought to result from a deficiency of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). This enzyme normally converts cortisol to inactive cortisone and is postulated to thus confer specificity for aldosterone upon the mineralocorticoid receptor. We have analysed the gene encoding the kidney isozyme of 11 beta HSD and found mutations on both alleles in nine of 11 AME patients (eight of nine kindreds). These mutations markedly affect enzymatic activity. They thus permit cortisol to occupy the renal mineralocorticoid receptor and thereby cause sodium retention and hypertension.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2003

Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2000

Association between a variant in the 11 beta-hydroxysteroid dehydrogenase type 2 gene and primary hypertension.

The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11BHSD2) converts cortisol to cortisone in the kidney, thereby protecting the mineralocorticoid receptor from the mineralocorticoid actions of cortisol. The syndrome of Apparent Mineralocorticoid Excess (AME), a rare monogenic form of early onset hypertension with autosomal recessive inheritance, is caused by homozygous or compound heterozygous loss of function mutations in the 11BHSD2 gene. Association has been reported between a microsatellite marker flanking the 11BHSD2 gene (D16S496) and primary hypertension. The aim of this study was to identify variants in the 11BHSD2 gene and to test if such variants or the D16S496 are associated with primary hypertension, in Swedes. To address this, the coding sequences of the 11BHSD2 gene was screened for mutations in 20 patients with primary hypertension with single strand conformation polymorphism and direct DNA sequencing techniques. A polymorphism was identified in exon 3; G534A (Glu178Glu). This polymorphism and the D16S496 microsatellite were tested for association with primary hypertension in a population consisting of 292 patients with primary hypertension and 263 normotensive control subjects. The frequency of G534G homozygotes was higher in patients with primary hypertension than in normotensive control subjects (92.8% vs 87.8%; P < 0.05). The allele frequencies of the D16S496 microsatellite did not differ between the two groups (chi(2) = 11.0, df = 10; P = 0.36). In conclusion, over-representation of individuals homozygous for the G534 allele in hypertensive patients compared with control subjects suggests that a mutation in linkage disequilibrium with the G534A polymorphism could increase susceptibility to primary hypertension. Journal of Human Hypertension (2000) 14, 819-823

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2003

Abnormal expression of 11 beta-hydroxysteroid dehydrogenase type 2 in human pituitary adenomas: a prereceptor determinant of pituitary cell proliferation.

The physiological effects of glucocorticoids (GCs) are, at least in part, mediated by inhibition of cell proliferation. Two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) interconvert cortisol (F) and inactive cortisone (E), and are thus able to modulate GC action at an autocrine level. Previously, we have demonstrated absent expression of 11 beta-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11 beta-HSD2 was readily demonstrable. Here we have used real-time RT-PCR to quantify expression of mRNA for 11 beta-HSD1 and 2 in 105 human pituitary tumors and have performed enzyme expression and activity studies in primary pituitary cultures. Overall, pituitary tumors expressed lower levels of 11 beta-HSDl mRNA compared with normals (0.2-fold, P<0.05). In contrast, expression of 11 beta-HSD2 mRNA was 9.8-fold greater in tumors than in normals (P<0.001). Enzyme assays showed significant 11 beta-HSD2 activity (71.9+/-22.3 pmol/h/mg protein (mean+/-s.d.)) but no detectable 11 beta-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11 beta-HSD2 inhibitor) resulted in a 30.3+/-7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11 beta-HSDl to 11 beta-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11 beta-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.

11-beta-Hydroxysteroid Dehydrogenases↗