Mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21) binds steroids differently from other aldo-keto reductases: identification and characterization of amino acid residues critical for substrate binding

The mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD) is the unique known member of the aldo-keto reductase (AKR) superfamily able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epi-testosterone (epi-T), the 17alpha-epimer of testosterone. Structural and mutagenic studies had already identified one of the residues delineating the steroid-binding cavity, A24, as the major molecular determinant for the stereospecificity of m17alpha-HSD. We report here a ternary complex crystal structure (m17alpha-HSD:NADP(+):epi-T) determined at 1.85 A resolution that confirms this and reveals a unique steroid-binding mode for an AKR enzyme. Indeed, in addition to the interactions found in all other AKRs (van der Waals contacts stabilizing the core of the steroid and the hydrogen bonds established at the catalytic site by the Y55 and H117 residues with the oxygen atom of the ketone group to be reduced), m17alpha-HSD establishes with the other extremity of the steroid nucleus an additional interaction involving K31. By combining direct mutagenesis and kinetic studies, we found that the elimination of this hydrogen bond did not affect the affinity of the enzyme for its steroid substrate but led to a slight but significant increase of its catalytic efficiency (k(cat)/K(m)), suggesting a role for K31 in the release of the steroidal product at the end of the reaction. This previously unobserved steroid-binding mode for an AKR is similar to that adopted by other steroid-binding proteins, the hydroxysteroid dehydrogenases of the short-chain dehydrogenases/reductases (SDR) family and the steroid hormone nuclear receptors. Mutagenesis and structural studies made on the human type 3 3alpha-HSD, a closely related enzyme that shares 73% amino acids identity with the m17alpha-HSD, also revealed that the residue at position 24 of these two enzymes directly affects the binding and/or the release of NADPH, in addition to its role in their 17alpha/17beta stereospecificity.     

Human 20alpha-hydroxysteroid dehydrogenase: crystallographic and site-directed mutagenesis studies lead to the identification of an alternative binding site for C21-steroids

Human 20alpha-hydroxysteroid dehydrogenase (h20alpha-HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20alpha-hydroxy-progesterone (20alpha-OHProg). Although h20alpha-HSD shares 98% sequence identity with human type 3 3alpha-HSD (h3alpha-HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20alpha-HSD in a ternary complex with NADP(+) and 20alpha-OHProg at 1.59A resolution. The steroid is stabilized by numerous hydrophobic interactions and a hydrogen bond between its O20 and the N(epsilon ) atom of His222. This new interaction prevents the formation of a hydrogen bond with the cofactor, as seen in h3alpha-HSD3 ternary complexes. By combining structural, direct mutagenesis and kinetic studies, we found that the H(222)I substitution decreases the K(m) value for the cofactor 95-fold. With these results, we hypothesize that the rotation of the lateral chain of His222 could be a mediating step between the transformation of Prog and the release of the cofactor. Moreover, crystal structure analysis and direct mutagenesis experiments lead us to identify a new residue involved in the binding of Prog. Indeed, the R(304)L substitution leads to a 65-fold decrease in the K(m) value for Prog reduction. We thus propose that Prog is maintained in a new steroid-binding site composed mainly of residues found in the carboxy-terminal region of the protein.     

Substrate specificity and inhibitor analyses of human steroid 5β-reductase (AKR1D1)

Human steroid 5β-reductase (aldo-keto reductase 1D1) catalyzes the stereospecific NADPH-dependent reduction of the C4-C5 double bond of Δ⁴-ketosteroids to yield an A/B cis-ring junction. This cis-configuration is crucial for bile acid biosynthesis and plays important roles in steroid metabolism. The biochemical properties of the enzyme have not been thoroughly studied and conflicting data have been reported, partially due to the lack of highly homogeneous protein. In the present study, we systematically determined the substrate specificity of homogeneous human recombinant AKR1D1 using C18, C19, C21, and C27 Δ⁴-ketosteroids and assessed the pH-rate dependence of the enzyme. Our results show that AKR1D1 proficiently reduced all the steroids tested at physiological pH, indicating AKR1D1 is the only enzyme necessary for all the 5β-steroid metabolites present in humans. Substrate inhibition was observed with C18 to C21 steroids provided that the C11 position was unsubstituted. This structure activity relationship can be explained by the existence of a small alternative substrate binding pocket revealed by the AKR1D1 crystal structure. Non-steroidal anti-inflammatory drugs which are potent inhibitors of the related AKR1C enzymes do not inhibit AKR1D1. By contrast chenodeoxycholate and ursodeoxycholate were found to be potent non-competitive inhibitors suggesting that bile-acids may regulate their own synthesis at the level of AKR1D1 inhibition.     

Molecular docking simulations of steroid substrates into human cytosolic hydroxysteroid dehydrogenases (AKR1C1 and AKR1C2): insights into positional and stereochemical preferences

AKR1C1 and AKR1C2 are human cytosolic hydroxysteroid dehydrogenases, which play pivotal roles in the metabolism and action of natural and synthetic steroid hormones. The two enzymes are highly homologous, and have distinct positional and stereochemical preferences with various substrates. We performed molecular docking simulations of three steroid substrates, including an androgen (5alpha-dihydrotestosterone, DHT), a progestin (progesterone, PRO), and a synthetic hormone ([7alpha,17alpha]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one or tibolone, TIB), into the active sites of the two enzymes. For each substrate and enzyme pair, the activity inferred by the "productive" docking models (in which the spatial arrangement of the steroid and the cofactor would permit a reaction) matched the experimentally observed positional and stereochemical outcome. These productive conformations were energetically and statistically favored except for TIB and PRO with AKR1C2, where experimentally strong substrate inhibition and low activity were observed, respectively. Results showed that (i) a 3-ketosteroid (DHT) and a 20-ketosteroid (PRO) were reduced by AKR1C1 since the carbonyl groups could occupy the same position by "backwards" binding of steroids; (ii) 3alpha-reduced (DHT) and 3beta-reduced (TIB) products were formed by AKR1C2 since the angular methyl groups of the steroids were inverted by "upside-down" binding of steroids; and (iii) the 3beta- and 3alpha-reduction of DHT by AKR1C1 and AKR1C2, respectively occurred since the steroids employed a "swinging" motion to present opposite faces to the cofactor. Favorable nonproductive modes were observed with all substrates in both enzymes in which the steroid was bound at a "near-entry" position and/or an "in-middle" position, which may influence the reaction coordinate.     

Selectivity and potency of the retroprogesterone dydrogesterone in vitro

Dydrogesterone is widely used for menstrual disorders, endometriosis, threatened and habitual abortion and postmenopausal hormone replacement therapy. Although progestins have a promiscuous nature, dydrogesterone does not have clinically relevant androgenic, estrogenic, glucocorticoid or mineralocorticoid activities. To date, systematic biochemical characterization of this progestin and its active main metabolite, 20α-dihydrodydrogesterone, has not been performed in comparison to progesterone. The objective of this study was to evaluate the selectivity and potential androgenic/antiandrogenic effects of dydrogesterone and its metabolite in comparison to progesterone and medroxyprogesterone acetate by analyzing their interference with AR signaling in vitro. We characterized dydrogesterone and its metabolite for their binding and transactivation of androgen and other steroid hormone receptors and for their potential inhibitory effects against androgen biosynthetic enzymes, 17β-hydroxysteroid dehydrogenase types 3 and 5 and 5α-reductase types 1 and 2. We found that dydrogesterone resembled progesterone mainly in its progestogenic effects and less in its androgenic, anti-androgenic, glucocorticoid and antiglucocorticoid effects; whereas, 20α-dihydrodydrogesterone showed reduced progestogenic potency with no androgenic, glucocorticoid and mineralocorticoid effects. Effects on the androgen and glucocorticoid receptor differed depending on the technology used to investigate transactivation. Progesterone, but not dydrogesterone and 20α-dihydrodydrogesterone, exerted anti-androgenic effects at the pre-receptor level by inhibiting 5α-reductase type 2. Dydrogesterone, 20α-dihydrodydrogesterone and progesterone inhibited the biosynthesis of testosterone catalyzed by 17β-hydroxysteroid dehydrogenase types 3 and 5; however, due to their micromolar K_i values, these activities appeared to be not of relevance at therapeutic levels. Overall, our data show that the anti-androgenic potential of dydrogesterone and 20α-dihydrodydrogesterone is less pronounced compared to progesterone.     

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol

Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD⁺-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD⁺ as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD⁺-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP⁺ specificity, suggesting their key roles in coenzyme specificity.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.     

The R453Q and D151A polymorphisms of hexose-6-phosphate dehydrogenase gene (H6PD) influence the polycystic ovary syndrome (PCOS) and obesity

Hexose-6-phosphate dehydrogenase (H6PDH) influences 11β-hydroxysteroid dehydrogenase activity, a key enzyme in the peripheral metabolism of cortisol that modulates insulin sensitivity in adipose tissue. To study the associations of R453Q and D151A polymorphisms in the H6PDH gene (H6PD) with polycystic ovary syndrome (PCOS) and their influence on clinical and metabolic variables, we genotyped 237 patients with PCOS and 135 control women for the R453Q (rs6688832) and D151A (rs34603401) variants in H6PD. The R453Q genotypes were distributed differently in patients and controls (χ(2)=9.55, P=0.002). Genotypes of D151A were distributed evenly in women with PCOS and controls, but showed a different distribution in non-obese and obese women (χ(2)=3.95, P=0.047), especially within the PCOS subgroup (χ(2)=4.65, P=0.031). A backward stepwise likelihood ratio logistic regression model (Nagelkerke's R(2)=0.490; χ(2)=164; P<0.0001) retained free testosterone (OR=1.13; 95% CI: 1.10-1.17) and H6PD Q453 alleles (OR=0.46; 95% CI: 0.27-0.79) as statistically significant predictors for PCOS, whereas homeostasis model assessment of insulin resistance and the H6PD D151A variant were excluded by the model. Both H6PD variants were associated with several phenotypic variables, including fasting insulin, homeostasis model assessment of insulin resistance and androstenedione levels. In summary, the R453Q and D151A variants of the H6PD gene are associated with PCOS and obesity, respectively, and may contribute to the PCOS phenotype by influencing obesity, insulin resistance and hyperandrogenism.     

Probing the inhibitor selectivity pocket of human 20α-hydroxysteroid dehydrogenase (AKR1C1) with X-ray crystallography and site-directed mutagenesis

Human 20α-hydroxysteroid dehydrogenase (AKR1C1) is an important drug target due to its role in the development of lung and endometrial cancers, premature birth and neuronal disorders. We report the crystal structure of AKR1C1 complexed with the first structure-based designed inhibitor 3-chloro-5-phenylsalicylic acid (K_i = 0.86 nM) bound in the active site. The binding of 3-chloro-5-phenylsalicylic acid to AKR1C1 resulted in a conformational change in the side chain of Phe311 to accommodate the bulky phenyl ring substituent at the 5-position of the inhibitor. The contributions of the nonconserved residues Leu54, Leu306, Leu308 and Phe311 to the binding were further investigated by site-directed mutagenesis, and the effects of the mutations on the K_i value were determined. The Leu54Val and Leu306Ala mutations resulted in 6- and 81-fold increases, respectively, in K_i values compared to the wild-type enzyme, while the remaining mutations had little or no effects.     

Crystal structures of the conserved tRNA-modifying enzyme GidA: implications for its interaction with MnmE and substrate

GidA is a flavin-adenine-dinucleotide (FAD)-binding protein that is conserved among bacteria and eucarya. Together with MnmE, it is involved in the addition of a carboxymethylaminomethyl group to the uridine base in the wobble position (nucleotide 34) of tRNAs that read split codon boxes. Here, we report the crystal structures of the GidA proteins from both Escherichia coli and Chlorobium tepidum. The structures show that the protein can be divided into three domains: a first FAD-binding domain showing the classical Rossmann fold, a second α/β domain inserted between two strands of the Rossmann fold, and an α-helical C-terminal domain. The domain inserted into the Rossmann fold displays structural similarity to the nicotinamide-adenine-dinucleotide-(phosphate)-binding domains of phenol hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, and, correspondingly, we show that GidA binds NADH with high specificity as an initial donor of electrons. GidA behaves as a homodimer in solution. As revealed by the crystal structures, homodimerization is mediated via both the FAD-binding domain and the NADH-binding domain. Finally, a large patch of highly conserved, positively charged residues on the surface of GidA leading to the FAD-binding site suggests a tRNA-binding surface. We propose a model for the interaction between GidA and MnmE, which is supported by site-directed mutagenesis. Our data suggest that this interaction is modulated and potentially regulated by the switch function of the G domain of MnmE.     

An alternate pathway for androgen regulation of brain function: activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5alpha-androstane, 3beta,17beta-diol (3beta-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3beta-Diol is an important modulator of the stress response mediated by the hypothalmo-pituitary-adrenal axis. Furthermore, the actions of 3beta-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Neurosteroid modulation of N-methyl-D-aspartate receptors: molecular mechanism and behavioral effects

Glutamate is the main neurotransmitter released at synapses in the central nervous system of vertebrates. Its excitatory role is mediated through activation of specific glutamatergic ionotropic receptors, among which the N-methyl-d-aspartate (NMDA) receptor subtype has attracted considerable attention in recent years. Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of NMDA receptors. Many pharmacological compounds have been identified that affect the activity of NMDA receptors, including neurosteroids. This review summarizes our knowledge about molecular mechanisms underlying the neurosteroid action at NMDA receptors as well as about the action of neurosteroids in animal models of human diseases.     

The pattern of glycosyl- and sulfotransferase activities in cancer cell lines: a predictor of individual cancer-associated distinct carbohydrate structures for the structural identification of signature glycans

Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.     

Modulation of NRF2 signaling pathway by nuclear receptors: Implications for cancer

Nuclear factor-erythroid 2 p45-related factor 2 (NRF2, also known as Nfe2l2) plays a critical role in regulating cellular defense against electrophilic and oxidative stress by activating the expression of an array of antioxidant response element-dependent genes. On one hand, NRF2 activators have been used in clinical trials for cancer prevention and the treatment of diseases associated with oxidative stress; on the other hand, constitutive activation of NRF2 in many types of tumors contributes to the survival and growth of cancer cells, as well as resistance to anticancer therapy. In this review, we provide an overview of the NRF2 signaling pathway and discuss its role in carcinogenesis. We also introduce the inhibition of NRF2 by nuclear receptors. Further, we address the biological significance of regulation of the NRF2 signaling pathway by nuclear receptors in health and disease. Finally, we discuss the possible impact of NRF2 inhibition by nuclear receptors on cancer therapy.     

Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions

A highly active inhibitor of the binding of estrone and estradiol-17β to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH₂₀ and thin layer chromatography : it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17β anti-bodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.