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Purification of 11 beta-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique.

ABSTRACT: 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) efficiently inactivates potent glucocorticoid hormones (cortisol and corticosterone), leaving aldosterone unmetabolized. Abundant 11 beta-HSD2 activity in human placenta plays a central role in controlling fetal glucocorticoid exposure, which if excessive is harmful and may predispose to low birth weight and hypertension in adulthood. Similar 11 beta-HSD2 activity in the distal nephron protects mineralocorticoid receptors from glucocorticoids and appears to be important in normal blood pressure control. We have purified human placental 11 beta-HSD2 16000-fold, to homogeneity, and determined over 100 residues of the internal amino acid sequence. Purification was assisted by a novel technique allowing highly specific (single spot on two-dimensional electrophoresis) photoaffinity labelling of active 11 beta-HSD2 in crude tissue extracts by its glucocorticoid substrates. This work reveals that 11 beta-HSD2 is a member of the short-chain alcohol dehydrogenase superfamily (apparent monomer M(r) approximately 40,000). It is a very basic (apparent pI = 9.1) intrinsic membrane protein, requiring as yet undefined membrane constituents for full stability. Affinity chromatography and affinity labelling studies suggest that 11 beta-HSD2 has a compulsory ordered mechanism, with NAD+ binding first, followed by a conformational change allowing glucocorticoid binding with high affinity.

SUBMITTER: Brown RW

PROVIDER: S-EPMC1217009 | biostudies-other | 1996 Feb

REPOSITORIES: biostudies-other

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Renal medullary 11 beta-hydroxysteroid dehydrogenase type 1 in Dahl salt-sensitive hypertension.

Project description:The Dahl salt-sensitive rat is a widely used model of human salt-sensitive forms of hypertension. The kidney plays an important role in the pathogenesis of Dahl salt-sensitive hypertension, but the molecular mechanisms involved remain a subject of intensive investigation. Gene expression profiling studies suggested that 11 beta-hydroxysteroid dehydrogenase type 1 might be dysregulated in the renal medulla of Dahl salt-sensitive rats. Additional analysis confirmed that renal medullary expression of 11 beta-hydroxysteroid dehydrogenase type 1 was downregulated by a high-salt diet in SS-13BN rats, a consomic rat strain with reduced blood pressure salt sensitivity, but not in Dahl salt-sensitive rats. 11 beta-Hydroxysteroid dehydrogenase type 1 is known to convert inactive 11-dehydrocorticosterone to active corticosterone. The urinary corticosterone/11-dehydrocorticosterone ratio as well as urinary excretion of corticosterone was higher in Dahl salt-sensitive rats than in SS-13BN rats. Knockdown of renal medullary 11 beta-hydroxysteroid dehydrogenase type 1 with small-interfering RNA attenuated the early phase of salt-induced hypertension in Dahl salt-sensitive rats and reduced urinary excretion of corticosterone. Knockdown of 11 beta-hydroxysteroid dehydrogenase type 1 did not affect blood pressure in SS-13BN rats. Long-term attenuation of salt-induced hypertension was achieved with small hairpin RNA targeting renal medullary 11 beta-hydroxysteroid dehydrogenase type 1. In summary, we have demonstrated that suppression of 11 beta-hydroxysteroid dehydrogenase type 1 expression in the renal medulla attenuates salt-induced hypertension in Dahl salt-sensitive rats.

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Functional effects of polymorphisms in the human gene encoding 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1): a sequence variant at the translation start of 11 beta-HSD1 alters enzyme levels.

Project description:Regeneration of active glucocorticoids within liver and adipose tissue by the enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) may be of pathophysiological importance in obesity and metabolic syndrome and is a therapeutic target in type 2 diabetes. Polymorphisms in HSD11B1, the gene encoding 11 beta-HSD1, have been associated with metabolic phenotype in humans, including type 2 diabetes and hypertension. Here, we have tested the functional consequences of two single nucleotide polymorphisms located in contexts that potentially affect tissue levels of 11 beta-HSD1. We report no effect of allelic variation at rs846910, a polymorphism within the 5'-flanking region of the gene on HSD11B1 promoter activity in vitro. However, compared with the common G allele, the A allele of rs13306421, a polymorphism located two nucleotides 5' to the translation initiation site, gave higher 11 beta-HSD1 expression and activity in vitro and was translated at higher levels in in vitro translation reactions, possibly associated with a lower frequency of "leaky scanning." These data suggest that this polymorphism may have direct functional consequences on levels of 11 beta-HSD1 enzyme activity in vivo. However, the rs13306421 A sequence variant originally reported in other ethnic groups may be of low prevalence because it was not detected in a population of 600 European Caucasian women.

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Hydroxysteroid 11-Beta Dehydrogenase 1 Overexpression with Copy-Number Gain and Missense Mutations in Primary Gastrointestinal Stromal Tumors.

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Hydroxysteroid 17-Beta Dehydrogenase Type 10 Disease in Siblings.

Project description:Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) deficiency (HSD10 disease) is a rare X-linked neurodegenerative condition caused by abnormalities in the HSD17B10 gene. A total of 10 mutations have been reported in the literature since 2000. Described phenotypes include a severe neonatal or progressive infantile form with hypotonia, choreoathetosis, seizures, cardiomyopathy, neurodegeneration, and death, as well as an attenuated form with variable regression. Here we present the second report of a c.194T>C (p.V65A) mutation in two half-brothers with a clinical phenotype characterized by neurodevelopmental delay, choreoathetosis, visual loss, cardiac findings, and behavioral abnormalities, with regressions now noted in the older sibling. Neither has experienced a metabolic crisis. Both of the siblings had normal tandem mass spectroscopy analysis of their newborn screening samples. The older brother's phenotype may be complicated by the presence of a 3q29 microduplication. Diagnosis requires a high index of suspicion, as the characteristic urine organic acid pattern may escape detection. The exact pathogenic mechanism of disease remains to be elucidated, but may involve the non-dehydrogenase functionalities of the HSD10 protein. Our report highlights clinical features of two patients with the less fulminant phenotype associated with a V65A mutation, compares the reported phenotypes to date, and reviews recent findings regarding the potential pathophysiology of this condition.Summary Sentence Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) disease (HSD10 disease) is a rare X-linked neurodegenerative condition with a variable clinical phenotype; diagnosis requires a high index of suspicion.

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Hydroxysteroid 17β-dehydrogenase 11 accumulation on lipid droplets promotes ethanol-induced cellular steatosis.

Project description:Lipid droplets (LDs) are fat-storing organelles enclosed by a phospholipid monolayer, which harbors membrane-associated proteins that regulate distinct LD functions. LD proteins are degraded by the ubiquitin-proteasome system (UPS) and/or by lysosomes. Because chronic ethanol (EtOH) consumption diminishes the hepatic functions of the UPS and lysosomes, we hypothesized that continuous EtOH consumption slows the breakdown of lipogenic LD proteins targeted for degradation, thereby causing LD accumulation. Here, we report that LDs from livers of EtOH-fed rats exhibited higher levels of polyubiquitylated-proteins, linked at either lysine 48 (directed to proteasome) or lysine 63 (directed to lysosomes) than LDs from pair-fed control rats. MS proteomics of LD proteins, immunoprecipitated with UB remnant motif antibody (K-ε-GG), identified 75 potential UB proteins, of which 20 were altered by chronic EtOH administration. Among these, hydroxysteroid 17β-dehydrogenase 11 (HSD17β11) was prominent. Immunoblot analyses of LD fractions revealed that EtOH administration enriched HSD17β11 localization to LDs. When we overexpressed HSD17β11 in EtOH-metabolizing VA-13 cells, the steroid dehydrogenase 11 became principally localized to LDs, resulting in elevated cellular triglycerides (TGs). Ethanol exposure augmented cellular TG, while HSD17β11 siRNA decreased both control and EtOH-induced TG accumulation. Remarkably, HSD17β11 overexpression lowered the LD localization of adipose triglyceride lipase. EtOH exposure further reduced this localization. Reactivation of proteasome activity in VA-13 cells blocked the EtOH-induced rises in both HSD17β11 and TGs. Our findings indicate that EtOH exposure blocks HSD17β11 degradation by inhibiting the UPS, thereby stabilizing HSD17β11 on LD membranes, to prevent lipolysis by adipose triglyceride lipase and promote cellular LD accumulation.

| S-EPMC10060109 | biostudies-literature

Cloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2.

Project description:By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11 beta-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11 beta-HSD2 activity which is NAD(+)-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4 +/- 1.5, 43.9 +/- 8.5 and 119 +/- 15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10-20 nM). Rabbit antisera recognizing 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370--383)-peptide-carrier conjugate. Recombinant 11 beta-HSD2, like native human placental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post-translational processing for activity. 11 beta-HSD2 mRNA (approximately 1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11 beta-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11 beta-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribution resembling that in the adult is established by this stage in human development.

| S-EPMC1216963 | biostudies-other

Mutations of key hydrophobic surface residues of 11 beta-hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system.

Project description:11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11 beta-HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C-terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered K(m) values for steroids and an unaltered or increased k(cat). Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild-type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme.

| S-EPMC2775222 | biostudies-literature

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