A novel 17beta-hydroxysteroid dehydrogenase in the fungus Cochliobolus lunatus: new insights into the evolution of steroid-hormone signalling.
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ABSTRACT:
SUBMITTER: Lanisnik Rizner T
PROVIDER: S-EPMC1219993 | biostudies-other | 1999 Feb
REPOSITORIES: biostudies-other
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Project description:17beta-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17beta-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17beta-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17beta-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7 A resolution. The space group was identified as I4(1)22, with unit-cell parameters a = b = 67.14, c = 266.77 A. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies.
| S-EPMC1978160 | biostudies-literature
Project description:Breast cancer (BC) is still one of the major issues in world health, especially for women, which necessitates innovative therapeutic strategies. In this study, we investigated the efficacy of retinoic acid derivatives as inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which plays a crucial role in the biosynthesis and metabolism of oestrogen and thereby influences the progression of BC and, the main objective of this investigation is to identify the possible drug candidate against BC through computational drug design approach including PASS prediction, molecular docking, ADMET profiling, molecular dynamics simulations (MD) and density functional theory (DFT) calculations. The result has reported that total eight derivatives with high binding affinity and promising pharmacokinetic properties among 115 derivatives. In particular, ligands 04 and 07 exhibited a higher binding affinity with values of -9.9 kcal/mol and -9.1 kcal/mol, respectively, than the standard drug epirubicin hydrochloride, which had a binding affinity of -8.2 kcal/mol. The stability of the ligand-protein complexes was further confirmed by MD simulations over a 100-ns trajectory, which included assessments of hydrogen bonds, root mean square deviation (RMSD), root mean square Fluctuation (RMSF), dynamic cross-correlation matric (DCCM) and principal component analysis. The study emphasizes the need for experimental validation to confirm the therapeutic utility of these compounds. This study enhances the computational search for new BC drugs and establishes a solid foundation for subsequent experimental and clinical research.
| S-EPMC11319393 | biostudies-literature
Project description:Background17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17beta-HSDcl is also a homodimer under native conditions.ResultsWe have investigated here which secondary structure elements are involved in the dimerization of 17beta-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the alphaE-helices interact with the Asp121, Glu117 and Asp187 residues from the alphaE and alphaF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17beta-HSDcl monomeric, while the mutant 17beta-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity.ConclusionWe have shown by site-directed mutagenesis and structure/function analysis that 17beta-HSDcl dimerization involves the alphaE and alphaF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.
| S-EPMC1326212 | biostudies-literature
Project description:Human 17beta-hydroxysteroid dehydrogenase type 1 (17?-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. Previously, we reported the first observation of the influence of the enzyme on endogenous estrogen-responsive gene expression. Here, we demonstrate the impact of 17?-HSD1 expression on the breast cancer cell proteome and investigate its role in cell migration.17?-HSD1 was stably transfected in MCF7 cells and the proteome of the generated cells overexpressing 17?-HSD1 (MCF7-17?HSD1 cells) was compared to that of the wild type MCF7 cells. Proteomics study was performed using two-dimensional gel electrophoresis followed by mass spectrometry analysis of differentially expressed protein spots. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the transcription of individual gene. The effect of 17?-HSD1 on MCF7 cell migration was verified by a wound-healing assay.Proteomic data demonstrate that the expression of more than 59 proteins is modulated following 17?-HSD1 overexpression. 17?-HSD1 regulates the expression of important genes and proteins that are relevant to cell growth control, such as BRCA2 and CDKN1A interacting protein (BCCIP) and proliferating cell nuclear antigen (PCNA) which are down- and upregulated in MCF7-17?HSD1 cells, respectively. RT-qPCR data reveal that 17?-HSD1 increases the mRNA levels of estrogen receptors (ER) alpha and beta by 171 and 120%, respectively, while decreasing that of the androgen receptor by 64%. Interestingly, 17?-HSD1 increases the mRNA transcript (by 3.6 times) and the protein expression of the metastasis suppressor gene nm23-H1 and the expression of the two enzymes are closely correlated. We have further shown that 17?-HSD1 expression is associated with an increase of MCF7 cell migration.In addition to the regulation of important genes, we have demonstrated for the first time that 17?-HSD1 increases breast cancer cell migration, in spite of its positive regulation of the antimetastatic gene NM23. This is also correlated to its stimulation of breast cancer cell growth, further confirming its targeting in ER positive breast cancer. The novel findings in this study suggest several directions for future research on the contribution of 17?-HSD1 to breast cancer progression and related treatment.
| S-EPMC3446355 | biostudies-literature
Project description:ContextIncreased androgen production is a primary feature of polycystic ovary syndrome (PCOS) and appears to be an inherited trait. The gene for the steroidogenic enzyme type 5 17beta hydroxysteroid dehydrogenase (HSD17B5) was implicated as a candidate for the hyperandrogenemia of PCOS by a previous study that demonstrated an association of a single nucleotide polymorphism (SNP) in the promoter of this gene with PCOS.ObjectiveThe objective of the study was to replicate the previous report of association between the HSD17B5 gene and PCOS risk by genotyping the promoter SNP (as well as other SNPs in the region to provide improved coverage of the gene) in a large, well-characterized cohort suitable for replication study.DesignWomen with and without PCOS were genotyped for five SNPs in HSD17B5. SNPs and haplotypes were determined and tested for association with PCOS risk and phenotypic markers of PCOS.SettingSubjects were recruited from the reproductive endocrinology clinic at the University of Alabama at Birmingham; controls were recruited from the surrounding community. Genotyping took place at Cedars-Sinai Medical Center in Los Angeles.ParticipantsParticipants included 287 white women with PCOS and 187 white controls.Main measurementsHSD17B5 genotype, PCOS risk, and testosterone levels were measured.ResultsNo SNP or haplotype was significantly associated with PCOS risk, testosterone, or any of the traits tested.ConclusionsThese data suggest that polymorphisms in the HSD17B5 gene are not associated with PCOS risk or elevated testosterone as previously reported.
| S-EPMC2190745 | biostudies-literature
Project description:BackgroundWe have recently discovered that human type 12 17beta-HSD (h17beta-HSD12), a homolog of type 3 17beta-HSD, is a new estrogen-specific 17beta-hydroxysteroid dehydrogenase involved in the production of estradiol (E2). To further characterize this estradiol-producing enzyme, we have isolated the corresponding cDNA in the cynomolgus monkey (Macaca fascicularis), characterized its enzymatic activities and performed cellular localization using in situ hybridization.ResultsUsing HEK-293 cells stably expressing Macaca fascicularis type 12 17beta-HSD (mf17beta-HSD12), we have found that the mf17beta-HSD12 catalyzes efficiently and selectively the transformation of El into E2, in analogy with the h17beta-HSD12. We have also quantified the mf17beta-HSD12 mRNA expression levels in a series of Macaca fascicularis tissues using Quantitative RealTime PCR. The Macaca fascicularis 17beta-HSD12 mRNA is widely expressed with the highest levels tissues found in the cerebellum, spleen and adrenal with moderate level observed in all the other examined, namely the testis, ovary, cerebral cortex, liver, heart, prostate, mammary gland, myometrium, endometrium, skin, muscle and pancreas. To gain knowledge about the cellular localization of the mf17beta-HSD12 mRNA expression, we performed in situ hybridization using a 35S-labeled cRNA probe. Strong labeling was observed in epithelial cells and stromal cells of the mammary gland. In the uterus, the labeling is detected in epithelial cells and stromal cells of the endometrium.ConclusionThese results strongly suggest that the Macaca fascicularis 17beta-HSD12 is an essential partner of aromatase in the biosynthesis of estradiol (E2). It strongly suggests that in the estradiol biosynthesis pathway, the step of 17-ketoreduction comes after the step of the aromatization (the aromatization of 4-androstendione to estrone followed by the conversion of estrone into estradiol by estrogen specific l7beta-HSDs) which is in contrast with the hypothesis suggesting that 4-androstenedione is converted to testosterone followed by the aromatization of testosterone.
| S-EPMC1800897 | biostudies-literature
Project description:Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17beta-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17beta-HSDs analyzed were observed. Especially, the rodent 17beta-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17beta-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.
| S-EPMC2882332 | biostudies-literature
Project description:BACKGROUND: The Abeta-binding alcohol dehydrogenase/17beta-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10) is an enzyme involved in pivotal metabolic processes and in the mitochondrial dysfunction seen in the Alzheimer's disease. Here we use comparative genomic analyses to study the evolution of the HADH2 gene encoding ABAD/HSD10 across several eukaryotic species. RESULTS: Both vertebrate and nematode HADH2 genes showed a six-exon/five-intron organization while those of the insects had a reduced and varied number of exons (two to three). Eutherian mammal HADH2 genes revealed some highly conserved noncoding regions, which may indicate the presence of functional elements, namely in the upstream region about 1 kb of the transcription start site and in the first part of intron 1. These regions were also conserved between Tetraodon and Fugu fishes. We identified a conserved alternative splicing event between human and dog, which have a nine amino acid deletion, causing the removal of the strand betaF. This strand is one of the seven strands that compose the core beta-sheet of the Rossman fold dinucleotide-binding motif characteristic of the short chain dehydrogenase/reductase (SDR) family members. However, the fact that the substrate binding cleft residues are retained and the existence of a shared variant between human and dog suggest that it might be functional. Molecular adaptation analyses across eutherian mammal orthologues revealed the existence of sites under positive selection, some of which being localized in the substrate-binding cleft and in the insertion 1 region on loop D (an important region for the Abeta-binding to the enzyme). Interestingly, a higher than expected number of nonsynonymous substitutions were observed between human/chimpanzee and orangutan, with six out of the seven amino acid replacements being under molecular adaptation (including three in loop D and one in the substrate binding loop). CONCLUSION: Our study revealed that HADH2 genes maintained a reasonable conserved organization across a large evolutionary distance. The conserved noncoding regions identified among mammals and between pufferfishes, the evidence of an alternative splicing variant conserved between human and dog, and the detection of positive selection across eutherian mammals, may be of importance for further research on ABAD/HSD10 function and its implication in the Alzheimer's disease.
| S-EPMC1559703 | biostudies-literature
Project description:The active estrogen estradiol (E2) stimulates breast cancer cell (BCC) growth, whereas the androgen dihydrotestosterone (DHT) has shown an antiproliferative effect. The principal product synthesized by the 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is E2, although we have demonstrated that the purified enzyme also inactivates DHT. However, the direct roles of 17beta-HSD1 in sex-hormone regulation and BCC proliferation have not been completely established. Here, we show that 17beta-HSD1 inhibition suppresses DHT catabolism by 19%, whereas knockdown of the gene expression increases the concentration of DHT by 41% in the T47D BCC line. The 17beta-HSD1/DHT complex crystal structure reveals that DHT binds in both normal and reverse modes, but the latter mode leading to O3 reduction is preferred with stronger interactions. Using RNA interference and an inhibitor of 17beta-HSD1, we demonstrate that 17beta-HSD1 expression is negatively correlated to DHT levels in BCC but positively correlated to estrone reduction, E2 levels, and cell proliferation. 17beta-HSD1 inhibition reduces DHT inactivation, increasing the antiproliferative effect by DHT in T47D cells after 8 d treatment. Thus, 17beta-HSD1 up-regulates BCC growth by a dual action on estradiol synthesis and DHT inactivation. We have further demonstrated that 17beta-HSD1 can enhance the E2-induced expression of the endogenous estrogen-responsive gene pS2, providing an important information regarding the modulation of the estrogen responsiveness by 17beta-HSD1 that may also contribute to BCC growth. These results strongly support the rationale for inhibiting 17beta-HSD1 in breast cancer therapy to eliminate estrogen activation via the sulfatase pathway while avoiding the deprivation of DHT.
| S-EPMC5417535 | biostudies-literature
Project description:The data presented here are related to the research article entitled "Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1" (J.A. Aka, E.L. Calvo, S.X. Lin, 2016) [1]. We evaluated the effect of the steroidal enzyme 17?-HSD1 and its product, the estrogenic hormone 17-beta-estradiol (E2), on gene transcription profile of breast cancer cells. RNA interference technique was used to knock down the 17?-HSD1 gene (HSD17B1) in the hormone-dependent breast cancer cell line T47D in steroid-deprived medium. Transfected cells were subsequently treated with E2, and microarray analyses (with three contrasts) were used to investigate (i) the effect of 17?-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition, (ii) the effect of E2 on breast cancer gene expression and (iii) if E2 affects gene regulation by 17?-HSD1. Functional enrichments of the differentially expressed genes were assessed using Ingenuity Pathway Analysis (IPA). Here, we showed data on 140 genes that are induced or repressed 1.5 time or higher (p < 0.05) in the HSD17B1-silenced and E2-treated T47D cells revealed by microarray analysis, and presented the 14 functional terms found in the cancer and in the cell death and survival categories revealed by the IPA biological function analysis. Data on IPA Canonical Pathway and network analyses is also presented. Further discussion on gene regulation by 17?-HSD1 and E2 is provided in the accompanying publication [1].
| S-EPMC5122694 | biostudies-literature