Per- and polyfluoroalkyl substances inhibit human and rat 17ß-hydroxysteroid dehydrogenase 1: Quantitative structure-activity relationship and molecular docking analysis.

Per- and polyfluoroalkyl (PFAS) substances are enduring industrial materials. 17ß-Hydroxysteroid dehydrogenase isoform 1 (17ß-HSD1) is an estrogen metabolizing enzyme, which transforms estrone into estradiol in human placenta and rat ovary. Whether PFAS inhibit 17ß-HSD1 and what the structure-activity relationship (SAR) remains unexplored. We screened 18 PFAS for inhibiting human and rat 17ß-HSD1 in microsomes and studied their SAR and mode of action(MOA). Of the 11 perfluorocarboxylic acids (PFCAs), C8-C14 PFCAs at a concentration of 100 µM substantially inhibited human 17ß-HSD1, with order of C11 (half-maximal inhibition concentration, IC50, 8.94 µM) > C10 (10.52 µM) > C12 (14.90 µM) > C13 (30.97 µM) > C9 (43.20 µM) > C14 (44.83 µM) > C8 (73.38 µM) > others. Of the 7 per- and poly-fluorosulfonic acids (PFSAs), the potency was C8S (IC50, 14.93 µM) > C7S (80.70 µM) > C6S (177.80 µM) > others. Of the PFCAs, C8-C14 PFCAs at 100 µM markedly reduced rat 17ß-HSD1 activity, with order of C11 (IC50, 9.11 µM) > C12 (14.30 µM) > C10 (18.24 µM) > C13 (25.61 µM) > C9 (67.96 µM) > C8 (204.39 µM) > others. Of the PFSAs, the potency was C8S (IC50, 37.19 µM) > C7S (49.38 µM) > others. In contrast to PFOS (C6S), the partially fluorinated compound 6:2 FTS with an equivalent number of carbon atoms demonstrated no inhibition of human and rat 17ß-HSD1 activity at a concentration of 100 µM. The inhibition of human and rat enzymes by PFAS followed a V-shaped trend from C4 to C14, with a nadir at C11. Moreover, human 17ß-HSD1 was more sensitive than rat enzyme. PFAS inhibited human and rat 17ß-HSD1 in a mixed mode. Docking analysis revealed that they bind to the NADPH and steroid binding site of both 17ß-HSD1 enzymes. The 3D quantitative SAR (3D-QSAR) showed that hydrophobic region, hydrogen bond acceptor and donor are key factors in binding to 17ß-HSD1 active sites. In conclusion, PFAS exhibit inhibitory effects on human and rat 17ß-HSD1 depending on factors such as carbon chain length, degree of fluorination, and the presence of carboxylic acid or sulfonic acid groups, with a notable V-shaped shift observed at C11.

Light-switchable diazocines as potential inhibitors of testosterone-synthesizing 17ß-hydroxysteroid dehydrogenase 3.

In patients with prostate carcinoma as well as in some other cancer types, the reduction of testosterone levels is desired because the hormone stimulates cancer cell growth. One molecular target for this goal is the inhibition of 17ß-hydroxysteroid dehydrogenase type 3 (17ßHSD3), which produces testosterone from its direct precursor androstenedione. Recent research in this field is trying to harness photopharmacological properties of certain compounds so that the inhibitory effect could be turned on and off by irradiation. Seven new light-switchable diazocines were investigated with regard to their inhibition of 17ßHSD3. For this purpose, transfected HEK-293 cells and isolated microsomes were treated with the substrate and the potential inhibitors with and without irradiation for an incubation period of 3 or 5 h. The amount of generated testosterone was measured by UHPLC and compared between samples and control as well as between irradiated and non-irradiated samples. There was no significant difference between samples with and without irradiation. However, four of the seven diazocines led to a significantly lower testosterone production both in cell and in microsome assays. In some of the irradiated samples, a partial destruction of the diazocines was observed, indicated by an additional UHPLC peak. However, the influence on the inhibition is negligible, because the majority of the substance remained intact. In conclusion, new inhibitors of 17ßHSD3 have been found, but so far without the feature of a light switch, since the configurational alteration of the diazocines by irradiation did not lead to a change in bioactivity. Further modification might help to find a light-switching molecule that inhibits only in one configuration.

Comparison of structure-activity relationship for bisphenol analogs in the inhibition of gonadal 3ß-hydroxysteroid dehydrogenases among human, rat, and mouse.

Bisphenol A (BPA) is a widely used plastic material and its potential endocrine disrupting effect has restricted its use and increasing use of BPA alternatives has raised health concerns. However, the effect of bisphenol alternatives on steroidogenesis remains unclear. The objective of this study was to compare inhibitory potencies of 10 BPA alternatives in the inhibition of gonadal 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in three species (human, rat and mouse). The inhibitory potency for human 3ß-HSD2, rat 3ß-HSD1, and mouse 3ß-HSD6 ranged from bisphenol FL (IC50, 3.32 µM for human, 5.19 µM for rat, and 3.26 µM for mouse) to bisphenol E, F, and thiodiphenol (ineffective at 100 µM). Most BPA alternatives were mixed inhibitors of gonadal 3ß-HSD and they dose-dependently inhibited progesterone formation in KGN cells. Molecular docking analysis showed that all BPA analogs bind to steroid and NAD+ active sites. Lipophilicity of BPA alternatives was inversely correlated with IC50 values. In conclusion, BPA alternatives mostly can inhibit gonadal 3ß-HSDs and lipophilicity determines their inhibitory strength.

Abnormal Brain Protein Abundance and Cross-tissue mRNA Expression in Amyotrophic Lateral Sclerosis.

Due to the limitations of the present risk genes in understanding the etiology of amyotrophic lateral sclerosis (ALS), it is necessary to find additional causative genes utilizing novel approaches. In this study, we conducted a two-stage proteome-wide association study (PWAS) using ALS genome-wide association study (GWAS) data (N = 152,268) and two distinct human brain protein quantitative trait loci (pQTL) datasets (ROSMAP N = 376 and Banner N = 152) to identify ALS risk genes and prioritized candidate genes with Mendelian randomization (MR) and Bayesian colocalization analysis. Next, we verified the aberrant expression of risk genes in multiple tissues, including lower motor neurons, skeletal muscle, and whole blood. Six ALS risk genes (SCFD1, SARM1, TMEM175, BCS1L, WIPI2, and DHRS11) were found during the PWAS discovery phase, and SARM1 and BCS1L were confirmed during the validation phase. The following MR (p = 2.10 × 10-7) and Bayesian colocalization analysis (ROSMAP PP4 = 0.999, Banner PP4 = 0.999) confirmed the causal association between SARM1 and ALS. Further differential expression analysis revealed that SARM1 was markedly downregulated in lower motor neurons (p = 7.64 × 10-3), skeletal muscle (p = 9.34 × 10-3), and whole blood (p = 1.94 × 10-3). Our findings identified some promising protein candidates for future investigation as therapeutic targets. The dysregulation of SARM1 in multiple tissues provides a new way to explain ALS pathology.

Characterization of eight types of 17ß-hydroxysteroid dehydrogenases from the Japanese sardine Sardinops melanostictus: The probable role of type 12a in ovarian estradiol synthesis.

17ß-hydroxysteroid dehydrogenases (Hsd17bs) play a critical role in sex steroid biosynthesis. Although multiple types of Hsd17b have been found in fish, there is limited research on their expression and function. Recently, we succeeded in identifying eight types of Hsd17b (types 3, 4, 7, 8, 10, 12a, 12b, and 14) by RNA sequencing in the Japanese sardine Sardinops melanostictus, a commercially important clupeoid fish; however, a homologous sequence of Hsd17b1, which catalyzes the key reaction of estradiol-17ß (E2) synthesis, was absent. Here, we aimed to identify the Hsd17b type that plays a major role in E2 synthesis during ovarian development in Japanese sardine. The cDNAs encoding those eight types of Hsd17b were cloned and sequenced. The expressions of hsd17b3, hsd17b12a, and hsd17b12b were higher in ovary than in testis. In particular, hsd17b12a was predominantly expressed in the ovary. Expression of hsd17b3, hsd17b4, hsd17b12a, and hsd17b12b in the ovary increased during ovarian development. The enzymatic activities of Hsd17b3, Hsd17b12a, and Hsd17b12b were evaluated by expressing their recombinants in human embryonic kidney 293T cells. Hsd17b12a and Hsd17b12b catalyzed the conversion of androstenedione (AD) to testosterone (T) and estrone (E1) to E2. The results of in vitro bioassays using sardine ovaries indicated that E2 is synthesized from pregnenolone via AD and T, but not E1. These results suggest that Hsd17b12a plays a major role in E2 synthesis in sardine ovary by catalyzing the conversion of AD to T.

Involvement of Type 10 17ß-Hydroxysteroid Dehydrogenase in the Pathogenesis of Infantile Neurodegeneration and Alzheimer's Disease.

Type 10 17ß-hydroxysteroid dehydrogenase (17ß-HSD10) is the HSD17B10 gene product playing an appreciable role in cognitive functions. It is the main hub of exercise-upregulated mitochondrial proteins and is involved in a variety of metabolic pathways including neurosteroid metabolism to regulate allopregnanolone homeostasis. Deacetylation of 17ß-HSD10 by sirtuins helps regulate its catalytic activities. 17ß-HSD10 may also play a critical role in the control of mitochondrial structure, morphology and dynamics by acting as a member of the Parkin/PINK1 pathway, and by binding to cyclophilin D to open mitochondrial permeability pore. 17ß-HSD10 also serves as a component of RNase P necessary for mitochondrial tRNA maturation. This dehydrogenase can bind with the Aß peptide thereby enhancing neurotoxicity to brain cells. Even in the absence of Aß, its quantitative and qualitative variations can result in neurodegeneration. Since elevated levels of 17ß-HSD10 were found in brain cells of Alzheimer's disease (AD) patients and mouse AD models, it is considered to be a key factor in AD pathogenesis. Since data underlying Aß-binding-alcohol dehydrogenase (ABAD) were not secured from reported experiments, ABAD appears to be a fabricated alternative term for the HSD17B10 gene product. Results of this study would encourage researchers to solve the question why elevated levels of 17ß-HSD10 are present in brains of AD patients and mouse AD models. Searching specific inhibitors of 17ß-HSD10 may find candidates to reduce senile neurodegeneration and open new approaches for the treatment of AD.

17ß-hydroxysteroid dehydrogenases in the progression of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) has become a worldwide epidemic and a major public health problem, with a prevalence of approximately 25%. The pathogenesis of NAFLD is complex and may be affected by the environment and susceptible genetic factors, resulting in a highly variable disease course and no approved drugs in the clinic. Notably, 17ß-hydroxysteroid dehydrogenase type 13 (HSD17B13), which belongs to the 17ß-hydroxysteroid dehydrogenase superfamily (HSD17Bs), is closely related to the clinical outcome of liver disease. HSD17Bs consists of fifteen members, most related to steroid and lipid metabolism, and may have the same biological function as HSD17B13. In this review, we highlight recent advances in basic research on the functional activities, major substrates, and key roles of HSD17Bs in the progression of NAFLD to develop innovative anti-NAFLD drugs targeting HSD17Bs.

Benzophenone-1 and -2 UV-filters potently inhibit human, rat, and mouse gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Benzophenone (BP) ultraviolet (UV) -filters have been widely used to prevent adverse effects of UV. Whether they can disrupt gonadal steroidogenesis remains unclear. Gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD) catalyse the conversion of pregnenolone to progesterone. This study explored the effect of 12 BPs on human, rat, and mouse 3ß-HSD isoforms, and analysed the structure-activity relationship (SAR) and underlying mechanisms. The inhibitory potency was BP-1 (IC50, 5.66 ± 0.95 µM) > BP-2 (5.84 ± 2.22 µM) > BP-6 (185.8 ± 115.2 µM) > BP3-BP12 on human KGN 3ß-HSD2, BP-2 (5.90 ± 1.02 µM) > BP-1 (7.55 ± 1.26 µM) > BP3-B12 on rat testicular 3ß-HSD1, and BP-1 (15.04 ± 5.20 µM) > BP-2 (22.64 ± 11.81 µM) > BP-6（125.1 ± 34.65 µM）> BP-7 (161.1 ± 102.4 µM) > other BPs on mouse testicular 3ß-HSD6. BP-1 is a mixed inhibitor of human, rat, and mouse 3ß-HSDs, and BP-2 is a mixed inhibitor of human and rat 3ß-HSDs and a noncompetitive inhibitor of mouse 3ß-HSD6. 4-Hydroxyl substitution in the benzene ring plays a key role in enhancing potency of inhibiting human, rat, and mouse gonadal 3ß-HSDs. BP-1 and BP-2 can penetrate human KGN cells to inhibit progesterone secretion at ≥ 10 µM. Docking analysis revealed that the 4-hydroxyl group of BP-1 and BP-2 forms hydrogen bonds with residue Ser123 of human 3ß-HSD2 and residue Asp127 of rat 3ß-HSD1. In conclusion, this study demonstrates that BP-1 and BP-2 are the most potent inhibitors of human, rat, and mouse gonadal 3ß-HSDs and that there is a significant SAR difference.

Molecular docking, 3D-QSAR and simulation studies for identifying pharmacophoric features of indole derivatives as 17ß-hydroxysteroid dehydrogenase type 5 (17ß-HSD5) inhibitors.

Excess of androgens leads to various diseases such as Poly-Cystic Ovarian Syndrome, Prostate Cancer, Hirsutism, Obesity and Acne. 17ß-Hydroxysteroid Dehydrogenase type 5 (17ß-HSD5) converts androstenedione into testosterone peripherally, thereby significantly contributing to the development of these diseases. Indole-bearing scaffolds are reported as potential 17ß-HSD5 inhibitors for the manifestation of diseases arising due to androgen excess. In the present work, we have extensively performed a combination of molecular docking, Gaussian field-based 3D-QSAR, Pharmacophore mapping and MD-simulation studies (100 ns) to identify the pharmacophoric features of indole-based compounds as potent 17ß-HSD5 inhibitors. Molecular simulation studies of the most potent compound in the binding pocket of enzyme revealed that the compound 11 was stable in the binding pocket and showed good binding affinity through interactions with various residues of active site pocket. The Molecular mechanics Generalized Born surface area continuum solvation (MM/GBSA) and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations revealed that the compound 11 possessed a free binding energy of -36.36 kcal/mol and -7.00 kcal/mol, respectively, which was better as compared to reference compound Desmethyl indomethacin (DES). The developed pharmacophore will be helpful to design novel indole-based molecules as potent 17ß-HSD5 inhibitors for the treatment of various androgenic disorders.Communicated by Ramaswamy H. Sarma.

Prioritization of Drug Targets for Neurodegenerative Diseases by Integrating Genetic and Proteomic Data From Brain and Blood.

BACKGROUND: Neurodegenerative diseases are among the most prevalent and devastating neurological disorders, with few effective prevention and treatment strategies. We aimed to integrate genetic and proteomic data to prioritize drug targets for neurodegenerative diseases. METHODS: We screened human proteomes through Mendelian randomization to identify causal mediators of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, frontotemporal dementia, and Lewy body dementia. For instruments, we used brain and blood protein quantitative trait loci identified from one genome-wide association study with 376 participants and another with 3301 participants, respectively. Causal associations were subsequently validated by sensitivity analyses and colocalization. The safety and druggability of identified targets were also evaluated. RESULTS: Our analyses showed targeting BIN1, GRN, and RET levels in blood as well as ACE, ICA1L, MAP1S, SLC20A2, and TOM1L2 levels in brain might reduce Alzheimer's disease risk, while ICA1L, SLC20A2, and TOM1L2 were not recommended as prioritized drugs due to the identified potential side effects. Brain CD38, DGKQ, GPNMB, and SEC23IP were candidate targets for Parkinson's disease. Among them, GPNMB was the most promising target for Parkinson's disease with their causal relationship evidenced by studies on both brain and blood tissues. Interventions targeting FCRL3, LMAN2, and MAPK3 in blood and DHRS11, FAM120B, SHMT1, and TSFM in brain might affect multiple sclerosis risk. The risk of amyotrophic lateral sclerosis might be reduced by medications targeting DHRS11, PSMB3, SARM1, and SCFD1 in brain. CONCLUSIONS: Our study prioritized 22 proteins as targets for neurodegenerative diseases and provided preliminary evidence for drug development. Further studies are warranted to validate these targets.

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

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 17ß-dehydrogenase 11 (HSD17ß11) was prominent. Immunoblot analyses of LD fractions revealed that EtOH administration enriched HSD17ß11 localization to LDs. When we overexpressed HSD17ß11 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 HSD17ß11 siRNA decreased both control and EtOH-induced TG accumulation. Remarkably, HSD17ß11 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 HSD17ß11 and TGs. Our findings indicate that EtOH exposure blocks HSD17ß11 degradation by inhibiting the UPS, thereby stabilizing HSD17ß11 on LD membranes, to prevent lipolysis by adipose triglyceride lipase and promote cellular LD accumulation.

New insights into the substrate inhibition of human 17ß-hydroxysteroid dehydrogenase type 1.

Human type 1 17ß-hydroxysteroid dehydrogenase (17ß-HSD1),a member of the short-chain dehydrogenase/reductase family, catalyzes the last step in the bioactivation of the most potent estrogen estradiol with high specificity and is thus involved in estrogen-dependent diseases. As an oxidoreductase, 17ß-HSD1 can utilize both triphosphate and diphosphate cofactors in reaction at the molecular level, but more specific with triphosphate cofactor. The NADPH is much higher than NADP+ in living cells leading to preliminary reduction action. The enzyme also showed substrate-induced inhibition unprecedented in other members of 17ß-HSDs. Our previous study elucidated the structural mechanism of substrate inhibition is due to the reversely bound estrone (E1) in the substrate-binding pocket of the enzyme resulting in a dead-end complex. However, the effect of the cofactor preference on the substrate inhibition of the enzyme is not yet clear. In the present study, we solved the ternary crystal structures of 17ß-HSD1 in complex with E1 and cofactor analog NAD+ . Combined with molecular dynamics simulation using the enzyme with NADH/NADPH and different oriented E1 (normally oriented, E1N; reversely oriented, E1R), such ternary structure provides a complete picture of enzyme-substrate-cofactor interactions. The results reveal that different cofactors and substrate binding mode affect the allosteric effect between the two subunits of the enzyme. And the results from MD simulations confirmed that His221 plays a key role in the formation of dead-end complex in NADPH complex, and the absence of stable interaction between His221 and E1R in the NADH complex should be the main reason for its lack of substrate inhibition.

Molecular mechanisms underlying the defects of two novel mutations in the HSD17B3 gene found in the Tunisian population.

17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) converts Δ4-androstene-3,17-dione (androstenedione) to testosterone. It is expressed almost exclusively in the testes and is essential for appropriate male sexual development. More than 70 mutations in the HSD17B3 gene that cause 17ß-HSD3 deficiency and result in 46,XY Disorders of Sex Development (46,XY DSD) have been reported. This study describes three novel Tunisian cases with mutations in HSD17B3. The first patient is homozygous for the previously reported mutation p.C206X. The inheritance of this mutation seemed to be independent of consanguineous marriage, which can be explained by its high frequency in the Tunisian population. The second patient has a novel splice site mutation in intron 6 at position c.490 -6 T > C. A splicing assay revealed a complete omission of exon 7 in the resulting HSD17B3 mRNA transcript. Skipping of exon 7 in HSD17B3 is predicted to cause a frame shift in exon 8 that affects the catalytic site and results in a truncation in exon 9, leading to an inactive enzyme. The third patient is homozygous for the novel missense mutation p.K202M, representing the first mutation identified in the catalytic tetrad of 17ß-HSD3. Site-directed mutagenesis and enzyme activity measurements revealed a completely abolished 17ß-HSD3 activity of the p.K202M mutant, despite unaffected protein expression, compared to the wild-type enzyme. Furthermore, the present study emphasizes the importance of genetic counselling, detabooization of 46,XY DSD, and a sensitization of the Tunisian population for the risks of consanguineous marriage.

New Insights into Testosterone Biosynthesis: Novel Observations from HSD17B3 Deficient Mice.

Androgens such as testosterone and dihydrotestosterone (DHT) are essential for male sexual development, masculinisation, and fertility. Testosterone is produced via the canonical androgen production pathway and is essential for normal masculinisation and testis function. Disruption to androgen production can result in disorders of sexual development (DSD). In the canonical pathway, 17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) is viewed as a critical enzyme in the production of testosterone, performing the final conversion required. HSD17B3 deficiency in humans is associated with DSD due to low testosterone concentration during development. Individuals with HSD17B3 mutations have poorly masculinised external genitalia that can appear as ambiguous or female, whilst having internal Wolffian structures and testes. Recent studies in mice deficient in HSD17B3 have made the surprising finding that testosterone production is maintained, male mice are masculinised and remain fertile, suggesting differences between mice and human testosterone production exist. We discuss the phenotypic differences observed and the possible other pathways and enzymes that could be contributing to testosterone production and male development. The identification of alternative testosterone synthesising enzymes could inform the development of novel therapies to endogenously regulate testosterone production in individuals with testosterone deficiency.

The Mitochondrial Enzyme 17ßHSD10 Modulates Ischemic and Amyloid-ß-Induced Stress in Primary Mouse Astrocytes.

Severe brain metabolic dysfunction and amyloid-ß accumulation are key hallmarks of Alzheimer's disease (AD). While astrocytes contribute to both pathologic mechanisms, the role of their mitochondria, which is essential for signaling and maintenance of these processes, has been largely understudied. The current work provides the first direct evidence that the mitochondrial metabolic switch 17ß-hydroxysteroid dehydrogenase type 10 (17ßHSD10) is expressed and active in murine astrocytes from different brain regions. While it is known that this protein is overexpressed in the brains of AD patients, we found that 17ßHSD10 is also upregulated in astrocytes exposed to amyloidogenic and ischemic stress. Importantly, such catalytic overexpression of 17ßHSD10 inhibits mitochondrial respiration during increased energy demand. This observation contrasts with what has been found in neuronal and cancer model systems, which suggests astrocyte-specific mechanisms mediated by the protein. Furthermore, the catalytic upregulation of the enzyme exacerbates astrocytic damage, reactive oxygen species (ROS) generation and mitochondrial network alterations during amyloidogenic stress. On the other hand, 17ßHSD10 inhibition through AG18051 counters most of these effects. In conclusion, our data represents novel insights into the role of astrocytic mitochondria in metabolic and amyloidogenic stress with implications of 17ßHSD10 in multiple neurodegenerative mechanisms.

Mechanism of 17ß-estradiol degradation by Rhodococcus equi via the 4,5-seco pathway and its key genes.

Steroid estrogens have been detected in oceans, rivers, lakes, groundwaters, soils, and even urban water supply systems, thereby inevitably imposing serious impacts on human health and ecological safety. Indeed, many estrogen-degrading bacterial strains and degradation pathways have been reported, with the 4,5-seco pathway being particularly important. However, few studies have evaluated the use of the 4,5-seco pathway by actinomycetes to degrade 17ß-estradiol (E2). In this study, 5 genes involved in E2 degradation were identified in the Rhodococcus equi DSSKP-R-001 (R-001) genome and then heterologously expressed to confirm their functions. The transformation of E2 with hsd17b14 reached 63.7% within 30 h, resulting in transformation into estrone (E1). Furthermore, we found that At1g12200-encoded flavin-binding monooxygenase (FMOAt1g12200) can transform E1 at a rate of 51.6% within 30 h and can transform E1 into 4-hydroxyestrone (4-OH E1). In addition, catA and hsaC genes were identified to further transform 4-OH E1 at a rate of 97-99%, and this reaction was accomplished by C-C cleavage at the C4 position of the A ring of 4-OH E1. This study represents the first report on the roles of these genes in estrogen degradation and provides new insights into the mechanisms of microbial estrogen metabolism and a better understanding of E2 degradation via the 4,5-seco pathway by actinomycetes.

MiR-31 targets HSD17B14 and FSHR, and miR-20b targets HSD17B14 to affect apoptosis and steroid hormone metabolism of porcine ovarian granulosa cells.

Porcine 17-hydroxysteroid dehydrogenase type 14 (HSD17B14) and FSH reporter (FSHR) genes play important roles in the metabolism of steroid hormones and the apoptosis of ovarian granulosa cells (GCs). Our bioinformatics analyses and the dual luciferase reporter assays indicated that porcine miR-20b and miR-31 target the 3'-UTR region of HSD17B14 gene, and miR-31 also targets the 3'-UTR region of FSHR gene. Overexpression of porcine HSD17B14 gene promoted the conversion from estradiol (E2) to estrone (E1) and increased the apoptosis of porcine GCs. Overexpression of miR-20b down-regulated the mRNA and protein expression level of HSD17B14 gene, decreased the concentration of progesterone (P4) and E1, increased E2, as well as reduced apoptosis of GCs. Moreover, overexpression of miR-31 also down-regulated the protein expression level of HSD17B14 gene, decreased the concentration of P4 and E1, and increased E2. However, miR-31 promoted apoptosis of GCs by targeting to the 3'-UTR of porcine FSHR gene. Taken together, we found that both porcine miR-20b and miR-31 target HSD17B14 gene, but miR-31 also targets FSHR gene to regulate the metabolism of steroid hormones and the apoptosis of porcine ovarian GCs. These findings expand the epigenetic regulatory mechanism of porcine miR-31 and miR-20b in ovarian GCs.

Association of genetic polymorphisms with local steroid metabolism in human benign breasts.

PURPOSE: Although alterations of concentrations in circulating steroids have been linked to single nucleotide polymorphisms (SNPs) of steroidogenic enzymes, we hypothesized that SNPs of such enzymes located within the breast affect local steroid concentrations more than products of such SNPs absorbed from the circulation. METHODS: Steroids (estradiol, estrone, testosterone, androstenedione, DHEA, DHEA sulfate, progesterone) in nipple aspirate fluid (NAF) were purified by HPLC and they along with serum steroids were quantified by immunoassays. Polymorphisms of the transporter SLCO2B1 and enzymes HSD3B1, CYP19A1, HSD17B12, AKR1C3, CYP1B1, and SRD5A1 were measured in white blood cell DNA. RESULTS: Steroid concentrations in NAF of subjects with homozygous minor genotypes differed from those with heterozygotes, i.e., SLCO2B1 (rs2851069) decreased DHEAS (p = 0.04), HSD17B12 (rs11555762) increased estradiol (p < 0.004), and CYP1B1 (rs1056836) decreased estradiol (p = 0.017) and increased progesterone (p = 0.05). Also, in serum, CYP19A1 (rs10046 and rs700518) both decreased testosterone (p = 0.02) and SRD5A1 increased androstenedione (p = 0.006). Steroids in subjects with major homozygotes did not differ from those with heterozygotes indicating recessive characteristics. CONCLUSIONS: In the breast, SNPs were associated with decreased uptake of DHEAS (SLCO2B1), increased estradiol concentrations through increased oxidoreductase activity (HSD17B12), or decreased estradiol concentrations by presumed formation of 4-hydroxyestradiol (CYP1B1). CYP19A1 was associated with decreased testosterone concentrations in serum but had no significant effect on estrogen or androgen concentrations within the breast. The hormone differences observed in NAF were not usually evident in serum, indicating the importance of assessing the effect of these SNPs within the breast.

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.

Glioblastoma cells express crucial enzymes involved in androgen synthesis: 3ß-hydroxysteroid dehydrogenase, 17-20α-hydroxylase, 17ß-hydroxysteroid dehydrogenase and 5α-reductase.

Glioblastoma (GB) is the most common and aggressive primary brain tumour in adult humans. Therapeutic resistance and tumour recurrence after surgical removal contribute to poor prognosis for glioblastoma patients. Men are known to be more likely than women to develop an aggressive form of GB, and differences in sex steroids have emerged as a leading explanation for this finding. Studies indicate that the metabolism and proliferation of GB-derived cells are increased by sex steroids, the expression of androgen receptors (ARs) and the synthesis of androgens and oestrogens, suggesting that these hormones have a role in the tumour pathogenesis. The expression of aromatase, the enzyme that converts androgens to oestrogens, has been reported in glial cells and GB cell lines. Thus, it was necessary to test whether the steroidogenic enzymes involved in androgen synthesis are expressed in GB cells. Therefore, here, we investigated the expression of four key enzymes involved in androgen synthesis in human-derived GB cells. U87 cells were cultured in Dulbecco's modified Eagle medium plus foetal bovine serum and antibiotics on slides for immunocytochemistry or immunofluorescence. U87, LN229 and C6 cells were also cultured in multi-well chambers to obtain proteins for Western blotting. We used primary antibodies against 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17α-hydroxilase/17,20-lyase (P450c17), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and 5α-reductase. Immunocytochemistry, and immunofluorescence results revealed that glioblastoma cells express 3ß-HSD, P450c17, 17ß-HSD and 5α-reductase proteins in their cytoplasm. Moreover, Western blot analyses revealed bands corresponding to the molecular weight of these four enzymes in the three GB cell lines. Thus, glioblastoma cells have the key enzymatic machinery necessary to synthesize androgens, and these enzymes might be useful targets for new therapeutic approaches.