[Show abstract][Hide abstract] ABSTRACT: By interconverting glucocorticoids, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11beta-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11beta-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys(5) by Ser in 11beta-HSD1, but not of the analogous Lys(4) by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu(25)/Glu(26) in 11beta-HSD1 and Asp(25) in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys(35)/Lys(36) and Glu(25)/Glu(26) on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11beta-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11beta-HSD1, facing the ER lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11beta-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11beta-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.
[Show abstract][Hide abstract] ABSTRACT: The renal 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 catalyzes the NAD(+)-dependent oxidation of the C11-alcohol on cortisol and corticosterone to yield inactive 11-ketosteroids. The lack of purified active enzyme complicates structure-function analyses of 11beta-HSD2. Here, we constructed a 3D-structural model of 11beta-HSD2, based on known 3D-structures of other short-chain dehydrogenases/reductases (SDR), and functionally analyzed 11beta-HSD2 mutants predicted to be involved in cofactor binding. Our 3D-model explains the preference for NAD(+) over NADP(+) by the coulombic repulsion between the adenosine ribose 2'-phosphate on NADP(+) and the carboxylate on Glu(115) and to steric hindrance with the side chain on Glu(115). Indeed, replacement of Glu(115) with serine or threonine, lacking repulsive charge and unfavorable steric interactions, showed only 3-fold preference for NAD(+), compared to 40-fold for wild-type 11beta-HSD2. Mutation of both Asp(91) and Glu(115) to serine raised NADP(+)-dependent activity to that with NAD(+), but caused reduced enzymatic activity. The 3D-model predicted that this is due to a loss of stabilizing interactions of Asp(91) with Cys(90), Glu(115), Asn(117) and Gly(120). Thus, predictions using the 3D-model combined with analysis of mutants allowed the identification of residues critical for NAD(+)-dependent activity of 11beta-HSD2.
[Show abstract][Hide abstract] ABSTRACT: 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 has been considered to protect the mineralocorticoid receptor (MR) by converting 11beta-hydroxyglucocorticoids into their inactive 11-keto forms, thereby providing specificity to the MR for aldosterone. To investigate the functional protection of the MR by 11beta-HSD2, we coexpressed epitope-tagged MR and 11beta-HSD2 in HEK-293 cells lacking 11beta-HSD2 activity and analyzed their subcellular localization by fluorescence microscopy. When expressed alone in the absence of hormones, the MR was both cytoplasmic and nuclear. However, when coexpressed with 11beta-HSD2, the MR displayed a reticular distribution pattern, suggesting association with 11beta-HSD2 at the endoplasmic reticulum membrane. The endoplasmic reticulum membrane localization of the MR was observed upon coexpression only with 11beta-HSD2, but not with 11beta-HSD1 or other steroid-metabolizing enzymes. Aldosterone induced rapid nuclear translocation of the MR, whereas moderate cortisol concentrations (10-200 nm) did not activate the receptor, due to 11beta-HSD2-dependent oxidation to cortisone. Compromised 11beta-HSD2 activity (due to genetic mutations, the presence of inhibitors, or saturating cortisol concentrations) led to cortisol-induced nuclear accumulation of the MR. Surprisingly, the 11beta-HSD2 product cortisone blocked the aldosterone-induced MR activation by a strictly 11beta-HSD2-dependent mechanism. Our results provide evidence that 11beta-HSD2, besides inactivating 11beta-hydroxyglucocorticoids, functionally interacts with the MR and directly regulates the magnitude of aldosterone-induced MR activation.
[Show abstract][Hide abstract] ABSTRACT: Renal 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) is an enzyme responsible for the peripheral inactivation of cortisol to cortisone in mineralocorticoid target tissues. Mutations in the gene encoding 11betaHSD2 cause the syndrome of apparent mineralocorticoid excess (AME), an autosomal recessive form of inherited hypertension, in which cortisol acts as a potent mineralocorticoid. The mutations reported to date have been confined to exons 3-5. Here, we describe two siblings, 1 and 2 yr old, who were diagnosed with hypokalemic hypertension and low plasma aldosterone and renin levels, indicating mineralocorticoid hypertension. Analysis of urinary steroid metabolites showed a markedly impaired metabolism of cortisol, with (tetrahydrocortisol + 5alpha-tetrahydrocortisol)/tetrahydrocortisone ratios of 40-60, and nearly absent urinary free cortisone. Although phenotypically normal, the heterozygous parents showed a disturbed cortisol metabolism. Genetic analysis of the HSD11B2 gene from the AME patients revealed the homozygous deletion of six nucleotides in exon 2 with the resultant loss of amino acids Leu(114) and Glu(115), representing the first alteration found in the cofactor-binding domain. The deletion mutant, expressed in HEK-293 cells, showed an approximately 20-fold lower maximum velocity but increased apparent affinity for cortisol and corticosterone. In contrast, two additionally constructed substitutions, Glu(115) to Gln or Lys, showed increased maximal velocity and apparent affinity for 11beta-hydroxyglucocorticoids. Functional analysis of wild-type and mutant proteins indicated that a disturbed conformation of the cofactor-binding domain, but not the missing negative charge of Glu(115), led to the observed decreased activity of the deletion mutant. Considered together, these findings provide evidence for a role of Glu(115) in determining cofactor-binding specificity of 11betaHSD2 and emphasize the importance of structure-function analysis to elucidate the molecular mechanism of AME.
[Show abstract][Hide abstract] ABSTRACT: 11beta-Hydroxysteroid dehydrogenase enzymes (11beta- HSD) regulate the ratio of active endogenous glucocorticoids to their inactive keto-metabolites, thereby controlling the access of glucocorticoids to their cognate receptors. In this study, the topology and intracellular localization of 11beta-HSD1 and 11beta-HSD2 have been analyzed by immunohistochemistry and protease protection assays of in vitro transcription/translation products. 11beta-HSD constructs, tagged with the FLAG epitope, were transiently expressed in HEK-293 cells. The enzymatic characteristics of tagged and native enzymes were indistinguishable. Fluorescence microscopy demonstrated the localization of both 11beta-HSD1 and 11beta-HSD2 exclusively to the endoplasmic reticulum (ER) membrane. To examine the orientation of tagged 11beta-HSD enzymes within the ER membrane, we stained selectively permeabilized HEK-293 cells with anti-FLAG antibody. Immunohistochemistry revealed that the N terminus of 11beta-HSD1 is cytoplasmic, and the catalytic domain containing the C terminus is protruding into the ER lumen. In contrast, the N terminus of 11beta-HSD2 is lumenal, and the catalytic domain is facing the cytoplasm. Chimeric proteins where the N-terminal anchor sequences of 11beta-HSD1 and 11beta-HSD2 were exchanged adopted inverted orientation in the ER membrane. However, both chimeric proteins were not catalytically active. Furthermore, mutation of a tyrosine motif to alanine in the transmembrane segment of 11beta-HSD1 significantly reduced V(max). The subcellular localization of 11beta-HSD1 was not affected by mutations of the tyrosine motif or of a di-lysine motif in the N terminus. However, residue Lys(5), but not Lys(6), turned out to be critical for the topology of 11beta-HSD1. Mutation of Lys(5) to Ser inverted the orientation of 11beta-HSD1 in the ER membrane without loss of catalytic activity. Our results emphasize the importance of the N-terminal transmembrane segments of 11beta-HSD enzymes for their proper function and demonstrate that they are sufficient to determine their orientation in the ER membrane.