Theoretical Analysis and Expression Profiling of 17ß-Hydroxysteroid Dehydrogenase Genes in Gonadal Development and Steroidogenesis of Leopard Coral Grouper (Plectropomus leopardus).

The differentiation and developmental trajectory of fish gonads, significantly important for fish breeding, culture, and production, has long been a focal point in the fields of fish genetics and developmental biology. However, the mechanism of gonadal differentiation in leopard coral grouper (Plectropomus leopardus) remains unclear. This study investigates the 17ß-Hydroxysteroid Dehydrogenase (Hsd17b) gene family in P. leopardus, with a focus on gene characterization, expression profiling, and functional analysis. The results reveal that the P. leopardus's Hsd17b gene family comprises 11 members, all belonging to the SDR superfamily. The amino acid similarity is only 12.96%, but conserved motifs, such as TGxxxGxG and S-Y-K, are present in these genes. Hsd17b12a and Hsd17b12b are unique homologs in fish, and chromosomal localization has confirmed that they are not derived from different transcripts of the same gene, but rather are two independent genes. The Hsd17b family genes, predominantly expressed in the liver, heart, gills, kidneys, and gonads, are involved in synthesizing or metabolizing sex steroid hormones and neurotransmitters, with their expression patterns during gonadal development categorized into three distinct categories. Notably, Hsd17b4 and Hsd17b12a were highly expressed in the testis and ovary, respectively, suggesting their involvement in the development of reproductive cells in these organs. Fluorescence in situ hybridization (FISH) further indicated specific expression sites for these genes, with Hsd17b4 primarily expressed in germ stem cells and Hsd17b12a in oocytes. This comprehensive study provides foundational insights into the role of the Hsd17b gene family in gonadal development and steroidogenesis in P. leopardus, contributing to the broader understanding of fish reproductive biology and aquaculture breeding.

[Study on reproductive toxicity of nano-cadmium sulfide with different particle sizes on male mice].

Objective: To investigate the reproductive toxicity of cadmium sulfide nanoparticles (Nano-CdS) with different particle sizes on male mice. Methods: In January 2019, 30 SPF grade male mice were randomly divided into a control group, an experimental group[CdS Ⅰ group (particle size approximately 5 nm), and a CdS Ⅱ group (particle size approximately 50 nm) ], with 10 mice in each group. The experimental group was orally gavaged with 100 mg/kg, once a day, while the control group was gavaged with an equal volume of physiological saline for 45 consecutive days. After 45 days, levels of cadmium accumulation in testis were determined directly by AAS, deformity and testicular histopathological changes were also observed. Serum testosterone levels were measured by enzyme-linked immunosorbentassay (ELISA), expression levels of P450scc, 17ß-HSD and P450c17 mRNA were determined by real-time PCR. P450c17 protein was determinated by Western Blot. Results: The histopathological results showed that the testes of the experimental group mice showed varying degrees of damage; Ultrastructural observation showed that the ultrastructure of mouse testicular cells in each experimental group showed varying degrees of mitochondrial expansion and disappearance of cristae, as well as irregular nuclear membranes. The degree of damage in CdS Ⅰ group was milder than that in CdS Ⅱ group. Compared with the control group, the cadmium content in the testes of the CdS Ⅰ and CdS Ⅱ groups significantly increased (P=0.001, 0.001), and the CdS Ⅱ group was higher than the CdS Ⅰ group (P=0.001). Compared with the control group, the levels of testosterone in the CdS Ⅰ and CdS Ⅱ groups decreased with statistical significance (P=0.001, 0.001). Real time fluorescence quantitative PCR results showed that compared with the control group, the experimental group's P450scc, 17ß-HSD. The expression levels of 17ß-HSD and P450c17 mRNA were significantly reduced, with statistically significant differences (P=0.001, 0.001, 0.001), and CdS Ⅱ group 17ß-HSD. The expression levels of 17ß-HSD and P450c17 mRNA were significantly lower than those of CdS Ⅰ group (P=0.001, 0.036). The Western Blot assay results showed that the expression levels of P450c17 protein in the testes of CdS Ⅰ and CdS Ⅱ groups of mice were significantly reduced, with statistical significance (P=0.001, 0.001) ; And the CdS Ⅱ group was significantly lower than the CdS Ⅰ group (P=0.001). According to Spearman correlation analysis, testosterone levels are correlated with P450scc, P450c17, 17ß-HSD mRNA. There is a highly positive correlation between 17ß-HSD mRNA levels, with statistically significant differences (r(s)=0.88, 0.80, 0.70, P=0.001, 0.001, 0.004) . Conclusion: Nano cadmium sulfide may induce reproductive toxicity by reducing the expression levels of key enzyme genes and enzyme protein activity in testosterone and its synthesis in mice, and the CdS Ⅱ group has a stronger toxic effect.

Efficient One-Step Biocatalytic Multienzyme Cascade Strategy for Direct Conversion of Phytosterol to C-17-Hydroxylated Steroids.

Steroidal 17-carbonyl reduction is crucial to the production of natural bioactive steroid medicines, and boldenone (BD) is one of the important C-17-hydroxylated steroids. Although efforts have been made to produce BD through biotransformation, the challenges of the complex transformation process, high substrate costs, and low catalytic efficiencies have yet to be mastered. Phytosterol (PS) is the most widely accepted substrate for the production of steroid medicines due to its similar foundational structure and ubiquitous sources. 17ß-Hydroxysteroid dehydrogenase (17ßHSD) and its native electron donor play significant roles in the 17ß-carbonyl reduction reaction of steroids. In this study, we bridged 17ßHSD with a cofactor regeneration strategy in Mycobacterium neoaurum to establish a one-step biocatalytic carbonyl reduction strategy for the efficient biosynthesis of BD from PS for the first time. After investigating different intracellular electron transfer strategies, we rationally designed the engineered strain with the coexpression of 17ßhsd and the glucose-6-phosphate dehydrogenase (G6PDH) gene in M. neoaurum. With the establishment of an intracellular cofactor regeneration strategy, the ratio of [NADPH]/[NADP+] was maintained at a relatively high level, the yield of BD increased from 17% (in MNR M3M-ayr1S.c) to 78% (in MNR M3M-ayr1&g6p with glucose supplementation), and the productivity was increased by 6.5-fold. Furthermore, under optimal glucose supplementation conditions, the yield of BD reached 82%, which is the highest yield reported for transformation from PS in one step. This study demonstrated an excellent strategy for the production of many other valuable carbonyl reduction steroidal products from natural inexpensive raw materials. IMPORTANCE Steroid C-17-carbonyl reduction is one of the important transformations for the production of valuable steroidal medicines or intermediates for the further synthesis of steroidal medicines, but it remains a challenge through either chemical or biological synthesis. Phytosterol can be obtained from low-cost residues of waste natural materials, and it is preferred as the economical and applicable substrate for steroid medicine production by Mycobacterium. This study explored a green and efficient one-step biocatalytic carbonyl reduction strategy for the direct conversion of phytosterol to C-17-hydroxylated steroids by bridging 17ß-hydroxysteroid dehydrogenase with a cofactor regeneration strategy in Mycobacterium neoaurum. This work has practical value for the production of many valuable hydroxylated steroids from natural inexpensive raw materials.

Impact of 17ß-HSD12, the 3-ketoacyl-CoA reductase of long-chain fatty acid synthesis, on breast cancer cell proliferation and migration.

Metabolic reprogramming of tumor cells involves upregulation of fatty acid (FA) synthesis to support high bioenergetic demands and membrane synthesis. This has been shown for cytosolic synthesis of FAs with up to 16 carbon atoms. Synthesis of long-chain fatty acids (LCFAs), including ω-6 and ω-3 polyunsaturated FAs, takes place at the endoplasmic reticulum. Despite increasing evidence for an important role of LCFAs in cancer, the impact of their synthesis in cancer cell growth has scarcely been studied. Here, we demonstrated that silencing of 17ß-hydroxysteroid dehydrogenase type 12 (17ß-HSD12), essentially catalyzing the 3-ketoacyl-CoA reduction step in LCFA production, modulates proliferation and migration of breast cancer cells in a cell line-dependent manner. Increased proliferation and migration after 17ß-HSD12 knockdown were partly mediated by metabolism of arachidonic acid towards COX2 and CYP1B1-derived eicosanoids. Decreased proliferation was rescued by increased glucose concentration and was preceded by reduced ATP production through oxidative phosphorylation and spare respiratory capacity. In addition, 17ß-HSD12 silencing was accompanied by alterations in unfolded protein response, including a decrease in CHOP expression and increase in eIF2α activation and the folding chaperone ERp44. Our study highlights the significance of LCFA biosynthesis for tumor cell physiology and unveils unknown aspects of breast cancer cell heterogeneity.

Coumarins as Tool Compounds to Aid the Discovery of Selective Function Modulators of Steroid Hormone Binding Proteins.

Steroid hormones play an essential role in a wide variety of actions in the body, such as in metabolism, inflammation, initiating and maintaining sexual differentiation and reproduction, immune functions, and stress response. Androgen, aromatase, and sulfatase pathway enzymes and nuclear receptors are responsible for steroid biosynthesis and sensing steroid hormones. Changes in steroid homeostasis are associated with many endocrine diseases. Thus, the discovery and development of novel drug candidates require a detailed understanding of the small molecule structure-activity relationship with enzymes and receptors participating in steroid hormone synthesis, signaling, and metabolism. Here, we show that simple coumarin derivatives can be employed to build cost-efficiently a set of molecules that derive essential features that enable easy discovery of selective and high-affinity molecules to target proteins. In addition, these compounds are also potent tool molecules to study the metabolism of any small molecule.

Friend or enemy? Review of 17ß-HSD10 and its role in human health or disease.

17ß-hydroxysteroid dehydrogenase (17ß-HSD10) is a multifunctional human enzyme with important roles both as a structural component and also as a catalyst of many metabolic pathways. This mitochondrial enzyme has important functions in the metabolism, development and aging of the neural system, where it is involved in the homeostasis of neurosteroids, especially in regard to estradiol, changes in which make it an essential part of neurodegenerative pathology. These roles therefore, indicate that 17ß-HSD10 may be a possible druggable target for neurodegenerative diseases including Alzheimer's disease (AD), and in hormone-dependent cancer. The objective of this review was to provide a summary about physiological functions and pathological roles of 17ß-HSD10 and the modulators of its activity.

17ß-hydroxysteroid dehydrogenase type 3 deficiency: female sex assignment and follow-up.

BACKGROUND: Deficiency of 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is a rare autosomal recessive 46,XY disorder of sex development (DSD). It is due to pathogenetic variants in the HSD17B3 gene. Mutated genes encode an abnormal enzyme with absent or reduced ability to convert Δ4-androstenedione (Δ4-A) to testosterone (T) in the fetal testis. Affected individuals are usually raised as females and diagnosis is made at puberty, when they show virilization. METHODS: A girl with a presumptive diagnosis of complete androgen insensitivity syndrome underwent endocrine and genetic assessment. Long-term follow-up was reported. RESULTS: The diagnosis of 17ß-HSD3 deficiency was made (stimulated T/Δ4-A ratio: 0.15; HSD17B3 gene analysis: c.277+4A>T in intron 3/c.640_645del (p.Glu214_Glu215del) in exon 9. After extensive information, parents decided to maintain female sex. Gonadal removal was performed and histological evaluation demonstrated deep fibrosis of testicular tissue. Follow-up till 8.5 years of age showed somatic and neuro-psychological development fitting with the female sex. CONCLUSIONS: Management of a child with the rare 17ß-HSD3 deficiency remains challenging. Any decision must be carefully evaluated with parents. Long-term follow-up must be warranted by a multidisciplinary DSD team to evaluate the adequacy of the choices made on quality of life in later life.

Benzothiazolyl Ureas are Low Micromolar and Uncompetitive Inhibitors of 17ß-HSD10 with Implications to Alzheimer's Disease Treatment.

Human 17ß-hydroxysteroid dehydrogenase type 10 is a multifunctional protein involved in many enzymatic and structural processes within mitochondria. This enzyme was suggested to be involved in several neurological diseases, e.g., mental retardation, Parkinson's disease, or Alzheimer's disease, in which it was shown to interact with the amyloid-beta peptide. We prepared approximately 60 new compounds based on a benzothiazolyl scaffold and evaluated their inhibitory ability and mechanism of action. The most potent inhibitors contained 3-chloro and 4-hydroxy substitution on the phenyl ring moiety, a small substituent at position 6 on the benzothiazole moiety, and the two moieties were connected via a urea linker (4at, 4bb, and 4bg). These compounds exhibited IC50 values of 1-2 µM and showed an uncompetitive mechanism of action with respect to the substrate, acetoacetyl-CoA. These uncompetitive benzothiazolyl inhibitors of 17ß-hydroxysteroid dehydrogenase type 10 are promising compounds for potential drugs for neurodegenerative diseases that warrant further research and development.

Ionic Environment Affects Biomolecular Interactions of Amyloid-ß: SPR Biosensor Study.

In early stages of Alzheimer's disease (AD), amyloid beta (Aß) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17ß-hydroxysteroid dehydrogenase 10 (17ß-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aß affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aß (Aß1-40, Aß1-42) with cypD and 17ß-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K+ and Mg2+ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aß1-40 and Aß1-42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aß1-40 to cypD and 17ß-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K+ and increased concentrations of Mg2+ promote the interaction of both mitochondrial proteins with Aß1-42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.

Blocking 17ß-hydroxysteroid dehydrogenase type 1 in endometrial cancer: a potential novel endocrine therapeutic approach.

The enzyme type 1 17ß-hydroxysteroid dehydrogenase (17ß-HSD-1), responsible for generating active 17ß-estradiol (E2) from low-active estrone (E1), is overexpressed in endometrial cancer (EC), thus implicating an increased intra-tissue generation of E2 in this estrogen-dependent condition. In this study, we explored the possibility of inhibiting 17ß-HSD-1 and impairing the generation of E2 from E1 in EC using in vitro, in vivo, and ex vivo models. We generated EC cell lines derived from the well-differentiated endometrial adenocarcinoma Ishikawa cell line and expressing levels of 17ß-HSD-1 similar to human tissues. In these cells, HPLC analysis showed that 17ß-HSD-1 activity could be blocked by a specific 17ß-HSD-1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by 17ß-HSD-1 inhibition. In vivo, tumors grafted on the chicken chorioallantoic membrane (CAM) demonstrated that E1 upregulated the expression of the estrogen responsive cyclin A similar to E2, which was impaired by 17ß-HSD-1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using 17ß-HSD-1-negative cells (negative control). Using a patient cohort of 52 primary ECs, we demonstrated the presence of 17ß-HSD-1 enzyme activity (ex vivo in tumor tissues, as measured by HPLC), which was inhibited by over 90% in more than 45% of ECs using the 17ß-HSD-1 inhibitor. Since drug treatment is generally indicated for metastatic/recurrent and not primary tumor, we next demonstrated the mRNA expression of the potential drug target, 17ß-HSD-1, in metastatic lesions using a second cohort of 37 EC patients. In conclusion, 17ß-HSD-1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and 17ß-HSD-1 inhibitor development to make candidate compounds suitable for the first human studies are awaited. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characterization of 17ß-hydroxysteroid dehydrogenase and regulators involved in estrogen degradation in Pseudomonas putida SJTE-1.

In bacteria, the enzyme catalyzing the transformation of 17ß-estradiol is considered the key enzyme for its metabolism, whose enzymatic activity and regulatory network influence the biodegradation efficiency of this typical estrogen. In this work, a novel 17ß-hydroxysteroid dehydrogenase (17ß-HSD) was characterized from the estrogen-degrading strain Pseudomonas putida SJTE-1, and two regulators were identified. This 17ß-HSD, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, could be induced by 17ß-estradiol and catalyzed the oxidization reaction at the C17 site of 17ß-estradiol efficiently. Its Km value was 0.068 mM, and its Vmax value was 56.26 µmol/min/mg; over 98% of 17ß-estradiol was oxidized into estrone in 5 min, indicating higher efficiency than other reported bacterial 17ß-HSDs. Furthermore, two genes (crgA and oxyR) adjacent to 17ß-hsd were studied which encoded the potential CrgA and OxyR regulators. Overexpression of crgA could enhance the transcription of 17ß-hsd, while that of oxyR resulted in the opposite effect. They could bind to the specific and different sites in the promoter region of 17ß-hsd gene directly, and binding of OxyR could be released by 17ß-estradiol. OxyR repressed the expression of 17ß-hsd by its specific binding to the conserved motif of GATA-N9-TATC, while CrgA activated the expression of this gene through its binding to the motif of T-N11-A. Therefore, this 17ß-HSD transformed 17ß-estradiol efficiently and the two regulators regulated its expression directly. This work could promote the study of the enzymatic mechanism and regulatory network of the estrogen biodegradation pathway in bacteria.

Novel Benzothiazole-based Ureas as 17ß-HSD10 Inhibitors, A Potential Alzheimer's Disease Treatment.

: It has long been established that mitochondrial dysfunction in Alzheimer's disease (AD) patients can trigger pathological changes in cell metabolism by altering metabolic enzymes such as the mitochondrial 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10), also known as amyloid-binding alcohol dehydrogenase (ABAD). We and others have shown that frentizole and riluzole derivatives can inhibit 17ß-HSD10 and that this inhibition is beneficial and holds therapeutic merit for the treatment of AD. Here we evaluate several novel series based on benzothiazolylurea scaffold evaluating key structural and activity relationships required for the inhibition of 17ß-HSD10. Results show that the most promising of these compounds have markedly increased potency on our previously published inhibitors, with the most promising exhibiting advantageous features like low cytotoxicity and target engagement in living cells.

One 3-oxoacyl-(acyl-Carrier-protein) reductase functions as 17ß-hydroxysteroid dehydrogenase in the estrogen-degrading Pseudomonas putida SJTE-1.

Pseudomonas putida SJTE-1 can utilize 17ß-estradiol (E2) as its carbon source, while the enzymes for E2 transformation in this strain is still unclear. 17ß-hydroxysteroid dehydrogenases (17ß-HSD) can catalyze the reduction/oxidation at C17 site of steroid hormone specifically, critical for steroid transformation. Here a novel 3-oxoacyl-(acyl-carrier protein) (ACP) reductase (ANI02794.1) was identified as it could bß-estradiol, and was proved to be capable of functioning as 17ß-HSD. Sequences alignment showed it contained the two consensus regions and the conserved residues of short-chain dehydrogenase/reductase (SDR). Its encoding gene was cloned and over-expressed in Escherichia coli BL21(DE3) strain, and the recombinant protein was purified by the metal-ion affinity chromatography with the yield of 18 mg/L culture. HPLC (High Performance Liquid Chromatography) detection showed this enzyme could convert 17ß-estradiol into estrone using NAD+ as cofactor. Its Km value was 0.082 mM and its Vmax value was 0.81 mM/s; its transformation efficiency of 17ß-estradiol into estrone was over 96.6% in five minutes. Its optimal temperature was 37 °C and optimal was pH 9.0; the divalent ions had different effects on the enzymatic activity. In conclusion, this 3-oxoacyl-ACP reductase functioned as 17ß-HSD in P. putida SJTE-1 and played important role in its estrogen metabolism.

17ß-Hydroxysteroid Dehydrogenase Type 2 Expression Is Induced by Androgen Signaling in Endometrial Cancer.

Endometrial cancer is one of the most common female pelvic cancers and has been considered an androgen-related malignancy. Several studies have demonstrated the anti-cell proliferative effect of androgen on endometrial cancer cells; however, the mechanisms of the anti-cancer effect of androgen remain largely unclear. 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2), which catalyzes the conversion of E2 to E1, is known to be upregulated by androgen treatment in breast cancer cells. In this study, we therefore focused on the role of androgen on estrogen dependence in endometrial cancer. Dihydrotestosterone (DHT) was found to induce 17ß-HSD2 mRNA and protein expression in HEC-1B endometrial cancer cells. DHT could also inhibit cell proliferation of HEC-1B when induced by estradiol treatment. In 19 endometrioid endometrial adenocarcinoma (EEA) tissues, intratumoral DHT concentration was measured by liquid chromatography/electrospray tandem mass spectrometry and was found to be significantly correlated with 17ß-HSD2 immunohistochemical status. We further examined the correlations between 17ß-HSD2 immunoreactivity and clinicopathological parameters in 53 EEA tissues. 17ß-HSD2 status was inversely associated with the histological grade, clinical stage, and cell proliferation marker Ki-67, and positively correlated with progesterone receptor expression. 17ß-HSD2 status tended to be positively associated with androgen receptor status. In 53 EEA cases, the 17ß-HSD2-positive group tended to have better prognosis than that for the negative group with respect to progression-free survival and endometrial cancer-specific survival. These findings suggest that androgen suppresses the estrogen dependence of endometrial cancer through the induction of 17ß-HSD2 in endometrial cancer.

Secretory expression, purification and functional characterization of 17ß-hydroxysteroid dehydrogenase type 1 from mammalian HEK293T cells.

17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) mainly catalyzes the reduction of estrone into estradiol. The enzymatic conversion is a critical step in estradiol accumulation in breast tissue, which is a valuable prognosis index of breast cancer disease. However, the source of 17ß-HSD1 for inhibitor design is limited. In this study, the fragment encoding human 17ß-HSD1 was successfully cloned and expressed in human embryonic kidney (HEK) 293T mammalian cells. The recombinant protein was purified by immobilized metal ion affinity chromatography yielding above 17 mg of purified 17ß-HSD1 protein per liter of cell culture, with a specific activity of 8.54 µmoL/min/mg of protein for conversion of estradiol into estrone, with NAD+ as cofactor at pH 9.2. Enzyme characterization studies revealed that the protein has estrogenic activity and the Km value for estrone is about 20 nM. The recombinant protein purified from transfected HEK293T cells had higher specific activity compared to that of the enzyme purified directly from placenta. The present data show that the mammalian cell expression system can provide active 17ß-HSD1 which is functionally identical to its natural counterpart and easy to purify in qualities suitable for its structure-function study.

Synthesis and evaluation of frentizole-based indolyl thiourea analogues as MAO/ABAD inhibitors for Alzheimer's disease treatment.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with an excessive accumulation of amyloid-beta peptide (Aß). Based on the multifactorial nature of AD, preparation of multi-target-directed ligands presents a viable option to address more pathological events at one time. A novel class of asymmetrical disubstituted indolyl thioureas have been designed and synthesized to interact with monoamine oxidase (MAO) and/or amyloid-binding alcohol dehydrogenase (ABAD). The design combines the features of known MAO inhibitors scaffolds (e.g. rasagiline or ladostigil) and a frentizole moiety with potential to interact with ABAD. Evaluation against MAO identified several compounds that inhibited in the low to moderate micromolar range. The most promising compound (19) inhibited human MAO-A and MAO-B with IC50 values of 6.34µM and 0.30µM, respectively. ABAD activity evaluation did not show any highly potent compound, but the compound series allowed identification of structural features to assist the future development of ABAD inhibitors. Finally, several of the compounds were found to be potent inhibitors of horseradish peroxidase (HRP), preventing the use of the Amplex™ Red assay to detect hydrogen peroxide produced by MAO, highlighting the need for serious precautions when using an enzyme-coupled assay.

Seasonal changes and sexual dimorphism in gene expression of StAR protein, steroidogenic enzymes and sex hormone receptors in the frog brain.

The brain of amphibians contains all the key enzymes of steroidogenesis and has a high steroidogenic activity. In seasonally-breeding amphibian species brain steroid levels fluctuate synchronously with the reproductive cycle. Here we report a study of gene expression of StAR protein, key steroidogenic enzymes and sex hormone receptors in the telencephalon (T) and diencephalon-mesencephalon (D-M) of male and female reproductive and post-reproductive Pelophylax esculentus, a seasonally breeding anuran amphibian. Significant differences in gene expression were observed between (a) the reproductive and post-reproductive phase, (b) the two brain regions and (c) male and female frogs. During the reproductive phase, star gene expression increased in the male (both T and D-M) but not in the female brain. Seasonal fluctuations in expression levels of hsd3b1, hsd17b1, srd5a1 and cyp19a1 genes for neurosteroidogenic enzymes occurred in D-M region of both sexes, with the higher levels in reproductive period. Moreover, the D-M region generally showed higher levels of gene expression than the T region in both sexes. Gene expression was higher in females than males for most genes, suggesting higher neurosteroid production in female brain. Seasonal and sex-linked changes were also observed in gene expression for androgen (ar) and estrogen (esr1, esr2) receptors, with the males showing the highest ar levels in reproductive phase and the highest esr1 and esr2 levels in post-reproductive phase; in contrast, females showed the maximum expression for all three genes in reproductive phase. The results are the first evidence for seasonal changes and sexual dimorphism of gene expression of the neurosteroidogenic pathway in amphibians.

Interference of Paraben Compounds with Estrogen Metabolism by Inhibition of 17ß-Hydroxysteroid Dehydrogenases.

Parabens are effective preservatives widely used in cosmetic products and processed food, with high human exposure. Recent evidence suggests that parabens exert estrogenic effects. This work investigated the potential interference of parabens with the estrogen-activating enzyme 17ß-hydroxysteroid dehydrogenase (17ß-HSD) 1 and the estrogen-inactivating 17ß-HSD2. A ligand-based 17ß-HSD2 pharmacophore model was applied to screen a cosmetic chemicals database, followed by in vitro testing of selected paraben compounds for inhibition of 17ß-HSD1 and 17ß-HSD2 activities. All tested parabens and paraben-like compounds, except their common metabolite p-hydroxybenzoic acid, inhibited 17ß-HSD2. Ethylparaben and ethyl vanillate inhibited 17ß-HSD2 with IC50 values of 4.6 ± 0.8 and 1.3 ± 0.3 µM, respectively. Additionally, parabens size-dependently inhibited 17ß-HSD1, whereby hexyl- and heptylparaben were most active with IC50 values of 2.6 ± 0.6 and 1.8 ± 0.3 µM. Low micromolar concentrations of hexyl- and heptylparaben decreased 17ß-HSD1 activity, and ethylparaben and ethyl vanillate decreased 17ß-HSD2 activity. However, regarding the very rapid metabolism of these compounds to the inactive p-hydroxybenzoic acid by esterases, it needs to be determined under which conditions low micromolar concentrations of these parabens or their mixtures can occur in target cells to effectively disturb estrogen effects in vivo.

Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11ß-hydroxysteroid dehydrogenase (11ß-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17ß-hydroxysteroid dehydrogenase (17ß-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17ß-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool.

Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17ß-hydroxysteroid dehydrogenase.

We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17ß-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3ß-hydroxysteroid dehydrogenase activity toward 5ß-androstanes, 5ß-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17ß-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.