Metabolism of a glutathione conjugate of 2-hydroxyoestradiol-17β in the adult male rat

Adult male rats with cannulated or ligated bile ducts were given S-(2-hydroxyoestradiol-1-yl)[(35)S]glutathione, S-(2-hydroxy[6,7-(3)H(2)]oestradiol-1-yl)glutathione or S-(2-hydroxyoestradiol-1-yl)[glycine-(3)H]glutathione by intraperitoneal injection. The recovery of radioactivity in the bile of bile duct-cannulated rats was 33-86% and in the urine of bile duct-ligated rats was 54-105%. Oestrogen thioether derivatives of glutathione, cysteinylglycine, cysteine and N-acetylcysteine were isolated from bile; only the N-acetylcysteine derivatives could be identified in the urine. The steroid moiety was characterized by microchemical tests before and after treatment with Raney nickel: 2-hydroxyoestradiol-17beta was released from the glutathione conjugate, and 2-hydroxyoestrone and 2-hydroxyoestrone 3-methyl ether from the other conjugates. From intact rats the recovery of administered radioactivity was about 15% in the urine and 5% in the faeces over a period of several days and the radioactivity appeared to be largely protein-bound. The results demonstrate that injected oestrogen-glutathione conjugate undergoes conversion into N-acetylcysteine derivatives in vivo. Oestrogen-glutathione conjugates formed in the intact rat may be excreted in an apparently non-steroidal, possibly protein-bound form, which would not be detected by current analytical techniques.     

Effect of progesterone pretreatment on the uptake of estradiol-17β by the uterine epithelium of the rat

Summary Progesterone pretreatment of ovariectomised rats resulted in a moderate (44%) lowering of the level of nuclear estradiol receptors found in the uterine epithelium 2 h after a single injection of this estrogen.     

17β-HSD2 inhibitors for the treatment of osteoporosis: Identification of a promising scaffold

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyses the conversion of active 17β-hydroxysteroids into the less active 17-ketosteroids thereby controlling the availability of biologically active estrogens (E2) and androgens (T) in the tissues. The skeletal disease osteoporosis occurs mainly in post-menopausal women and in elderly men when the levels of estrogens and androgens, respectively, decrease. Since 17β-HSD2 is present in osteoblasts, inhibition of this enzyme may provide a new and promising approach to prevent the onset of osteoporosis, keeping a certain level in estrogens and androgens in bone cells of ageing people. Hydroxynaphthyl, hydroxyphenyl and hydroxymethylphenyl-substituted moieties were synthesised as mimetics of the steroidal substrate. Compound 8 has been identified as promising scaffold for 17β-HSD2 inhibitors displaying high activity and good selectivity toward 17β-HSD1, ERα and ERβ.     

Neuroprotective Actions of the Synthetic Estrogen 17α-Ethynylestradiol in the Hippocampus

17α-Ethynylestradiol (EE2), a major constituent of many oral contraceptives, is similar in structure to 17β-estradiol, which has neuroprotective properties in several animal models. This study explored the potential neuroprotective actions of EE2 against kainic and quinolinic acid toxicity in the hippocampus of adult ovariectomized Wistar rats. A decrease in the number of Nissl-stained neurons and the induction of vimentin immunoreactivity in astrocytes was observed in the hilus of the dentate gyrus of the hippocampus after the administration of either kainic acid or quinolinic acid. EE2 prevented the neuronal loss and the induction of vimentin immunoreactivity induced by kainic acid at low (1 μg/rat) and high (10–100 μg/rat) doses and exerted a protection against quinolinic acid toxicity at a low dose (1 μg/rat) only. These observations demonstrate that EE2 exerts neuroprotective actions against excitotoxic insults. This finding is relevant for the design of new neuroprotective estrogenic compounds.     

Modeling the Role of TGF-β in Regulation of the Th17 Phenotype in the LPS-Driven Immune System

Airway exposure levels of lipopolysaccharide (LPS) are known to determine type I versus type II helper T cell induced experimental asthma. While low doses of LPS derive Th2 inflammatory responses, high (and/or intermediate) LPS levels induce Th1- or Th17-dominant responses. The present paper develops a mathematical model of the phenotypic switches among three Th phenotypes (Th1, Th2, and Th17) in response to various LPS levels. In the present work, we simplify the complex network of the interactions between cells and regulatory molecules. The model describes the nonlinear cross-talks between the IL-4/Th2 activities and a key regulatory molecule, transforming growth factor β (TGF-β), in response to high, intermediate, and low levels of LPS. The model characterizes development of three phenotypes (Th1, Th2, and Th17) and predicts the onset of a new phenotype, Th17, under the tight control of TGF-β. Analysis of the model illustrates the mono-, bi-, and oneway-switches in the key regulatory parameter sets in the absence or presence of time delays. The model also predicts coexistence of those phenotypes and Th1- or Th2-dominant immune responses in a spatial domain under various biochemical and bio-mechanical conditions in the microenvironment.     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.     

Quantitative analysis of estradiol-17β-induced changes in the ultrastructure of the liver of the male zebrafish,Brachydanio rerio

Summary Hepatocytes of male zebrafish, Brachydanio rerio, were studied by means of light- and electron-microscopy, following a period of maximally 16 days of in-vivo treatment with estradiol-17. The responsiveness of the male hepatocytes to this female sex steroid was investigated by use of morphometric methods. The results of this investigation show that the responsiveness was most obvious between 2 and 16 days, as revealed by an increase in cell size, accompanied by a proliferation of the granular endoplasmic reticulum and the Golgi complex. In addition, accumulations of glycogen granules, which are characteristic of hepatocytes in untreated males, had disappeared and lipid droplets had accumulated. These experimentally induced changes in the morphology of the male hepatocyte closely resemble those described for the female hepatocyte during the sexual cycle. It is concluded that the hepatocytes of male zebrafish can be stimulated by estradiol-17 to produce vitellogenin and that in female zebrafish this steroid is a key sex hormone responsible for vitellogenin production by the liver during the natural sexual cycle.     

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein

ABSTRACT

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP^C) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP^Sc) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP^C proteins have provided evidence of a conformational equilibrium between a 3₁₀-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165–175). In vitro cell-free experiments of the seeded conversion of PrP^C indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3₁₀-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3₁₀-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.     

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca2+ Channels

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (Ca_V) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP₂). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of Ca_V channels by PIP₂. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP₂ sensitivity of Ca_V2.2 and Ca_V1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.     

Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.     

The dimerization of glucagon-like peptide-2 MIMETIBODY™ is linked to leucine-17 in the glucagon-like peptide-2 region

Glucagon-like peptide-2 (GLP-2) is a member of the glucagon multigene family that is produced by intestinal enteroendocrine cells in response to food intake. GLP-2 stimulates growth of the intestinal epithelium, enhances its barrier functions, and increases nutrient uptake. Therefore, a GLP-2 agonist may be efficacious in human diseases characterized by malabsorption or injury to the gastrointestinal epithelium. MIMETIBODY? refers to a proprietary scaffold developed to extend the half-life of rapidly cleared peptides. It consists of a peptide linked to a scaffold that contains sequence elements from a human immunoglobulin G including those that allow recycling through the FcRn. The GLP-2 sequence was engineered into the MIMETIBODY? scaffold. The primary state of both GLP-2 and the GLP-2 MIMETIBODY? in DPBS was a noncovalently associated dimer indicative of self-interaction. The increased heterogeneity and the decreased lot-to-lot reproducibility caused by the self-interaction of therapeutic proteins are a challenge to drug development. A similar protein, GLP-1 MIMETIBODY?, contains the related GLP-1 peptide and does not form a dimer under similar conditions. Therefore, to minimize or abrogate dimerization, several variants were made by substituting GLP-2 amino acids with the corresponding amino acids from GLP-1. Molecular weight and secondary structure analyses reveal that substituting leucine for glutamine at position 17 (L17Q) reduces dimerization and α-helix content yet retains bioactivity.     

The effect of 17-N substituents on the activity of the opioid κ receptor in nalfurafine derivatives

We have previously reported the essential structure of the opioid κ receptor agonist nalfurafine hydrochloride (TRK-820) for binding to the κ receptor. In the course of this study, we focused on the effect of the substituent at 17-N in nalfurafine on the binding affinity for the κ receptor. The exchange of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups, which are strong electron withdrawing substituents, almost completely diminished the binding affinities for the μ and δ opioid receptors, but the binding affinity for the κ receptor was still maintained. As a result, nalfurafine derivatives with 17-fluoro-substituted alkyl groups showed higher selectivities for the κ receptor than did nalfurafine itself. With regard to the κ agonistic activities, the conversion of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups led to the gradual decrease of the agonistic activities in the order corresponding to their binding affinities for the κ receptor. In contrast, the derivative with the bulky 17-isobutyl group showed lower affinity and agonistic activity for the κ receptor than the derivatives with the smaller functional groups. This research suggested that both the electronic property and the steric characteristics of the 17-N substituent would have a great influence on the binding property for the κ receptor.     

Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17β-dehydrogenase

Human placental estradiol 17β-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one, a known substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 μM 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17β. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17β most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 βM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.     

Chemical synthesis of the 17-propanamide derivatives of stereoisomeric Δ14-17α- and 17β-estradiols: potential 17β-hydroxysteroid dehydrogenase inhibitors

The 17-propanamide derivatives of diastereomeric Δ¹⁴-17α- and 17β-estradiols, the potential candidates of a 17β-hydroxysteroid dehydrogenase (17β-HSD) inhibitor, were synthesized in 11 steps from estrone. The principal reactions employed involved in (1) conversion of estrone to the corresponding Δ¹⁴-estrone, (2) Grignard reaction of Δ¹⁴-estrone with allylmagnesium bromide followed by regioselective hydroboration of the resulting stereoisomeric 17ξ-allyl-Δ¹⁴-17ξ-ols with 9-borabicyclo[3.3.1]nonane (9-BBN), and (3) direct amidation of the 17ξ-O-/17ξ-C-spiro-γ-lactones with NH₃ under positive pressure of H₂.     

Antagonism of the actions of estrogens,androgens and progesterone by anordrin (2α,17α-diethynyl-A-nor-5α-androstane-2β, 17β-diol dipropionate)

Anordrin, an antifertility agent that is an antiestrogen with weak estrogenic activity, has been studied to further characterize its hormonal activities. A dose of 2.0 μg/mouse·day for 7 days did not increase the uterine content of protein, but it did inhibit to a small extent the effect of administered estradiol-17β on uterine protein content and more significantly the effect of estradiol-17β on the uterine content of progesterone receptors. Anordrin also decreased serum corticosteroid-binding globulin levels. Administration of an average daily dose of 160 μg/day of anordrin to intact male mice had no effect on weights of kidney, testis, or seminal vesicle after 10 days, but seminal vesicle weight was significantly decreased after 30 days at a slightly lower dose. Similarly, anordrin inhibited the increase in seminal vesicle weight induced by testosterone propionate treatment of castrated mice. In female mice anordrin failed to maintain deciduomata and blocked the ability of progesterone (2.0 mg/mouse·day) to do so. However, anordrin did not compete with the androgen [³H]R1881 for binding in kidney cytosol or with the progestin [³H]R5020 for uterine receptor sites. Anordrin also did not compete with [³H]corticosterone for binding to serum proteins.     

17β-estradiol inhibits the production of infectious particles of hepatitis C virus

Persistent infection with hepatitis C virus causes serious liver diseases, such as chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. The male gender is one of the critical factors in progression of hepatic fibrosis due to chronic HCV infection; thus female hormones may play a role in delaying the progression of hepatic fibrosis. It has also been reported that women are more likely than men to clear HCV in the acute phase of infection. These observations lead the present authors to the question: do female hormones inhibit HCV infection? In this study using HCV J6/JFH1 and Huh‐7.5 cells, the possible inhibitory effect(s) of female hormones such as 17β‐estradiol (the most potent physiological estrogen) and progesterone on HCV RNA replication, HCV protein synthesis and production of HCV infectious particles (virions) were analyzed. It was found that E₂, but not P₄, significantly inhibited production of the HCV virion without inhibiting HCV RNA replication or HCV protein synthesis. E₂–mediated inhibition of HCV virion production was abolished by a nuclear estrogen receptor (ER) antagonist ICI182780. Moreover, treatment with the ERα‐selective agonist 4, 4′, 4″‐ (4‐propyl‐[1H]‐pyrazole‐1, 3, 5‐triyl)trisphenol (PPT), but not with the ERβ‐selective agonist 2, 3‐bis (4‐hydroxyphenyl)‐propionitrile (DPN) or the G protein‐coupled receptor 30 (GPR30)‐selective agonist 1‐(4‐[6‐bromobenzo 1, 3 dioxol‐5‐yl]‐3a, 4, 5, 9b‐tetrahydro‐3H‐cyclopenta [c] quinolin‐8‐yl)‐ethanone (G‐1), significantly inhibited HCV virion production. Taken together, the present results suggest that the most potent physiological estrogen, E₂, inhibits the production of HCV infectious particles in an ERα–dependent manner.     

Solid-state NMR analysis of steroidal conformation of 17α- and 17β-estradiol in the absence and presence of lipid environment

Solid-state {(1)H}(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to 17β-estradiol (E2) and 17α-estradiol (E2α), to analyze the steroidal ring conformations of the two isomers in the absence and presence of lipids at the atomic level. In the absence of lipid, the high-resolution (13)C NMR signals of E2 in a powdered form show only singlet patterns, suggesting a single ring conformation. In contrast, the (13)C signals of E2α reveal multiplet patterns with splittings of 20-300Hz, implying multiple ring conformations. In the presence of a mimic of the lipid environment, made by mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, E2 and E2α revealed multiplet patterns different from those seen in the absence of lipids, indicating that the two isomers adopt multiple conformations in the lipid environment. In this work, on the basis of chemical shift isotropy and anisotropy analysis, we demonstrated that E2 and E2α prefer to adopt multiple steroidal ring conformations in the presence of a lipid environment, distinct from that observed in solution phase and powdered form.     

The role of estradiol 17β in the activation of the uterus during premature labor and the effect of naproxen,an inhibitor of prostaglandin synthesis

Fifty-two pregnant rats were ovariectomized on day 16 of gestation to induce estrogen and progesterone deficiencies and the animals were divided into four Groups. Ovariectomy alone (Group A) resulted in the premature delivery of 21% of the fetuses. When ovariectomy was followed by estrogen treatment restoring normal estrogen levels (Group B), premature delivery of the fetuses increased to 96%. Daily injections of 25mg/kg b.w. Naproxen (Group C), given from the day of ovariectomy to reduce prostaglandin synthesis, completely prevented premature delivery if the animals received no estradiol treatment and reduced prematurity to 50% if estradiol had been administered (Group D).It is concluded that the estrogen and progesterone deficiency, induced by ovariectomy, provokes a regulatory imbalance which promotes premature delivery. This imbalance is enhanced when the estradiol levels are restored to normal values, probably because estradiol increases the synthesis of prostaglandin, the intrinsic myometrial stimulant. Naproxen, an inhibitor of prostaglandin synthesis, restores the regulatory balance, partially or completely, depending on the estrogen levels.