Quantitative Characterization of Ectopic Adrenal Gene Expression in Fetal Testes in 21-Hydroxylase Deficient Mice.

Testicular adrenal rest tumors (TART) are a frequent and fertility impairing long-term complication in males with classic congenital adrenal hyperplasia. Due to lack of clear experimental data on their origin, they are hypothesized to be derived from ectopic adrenocortical cells within testicular tissue mainly growing upon stimulation by chronically elevated levels of adrenocorticotropin (ACTH). Alternatively, a more totipotent embryological origin has been discussed as the potential source of these tumors. The aim of this study was to quantify alterations of ectopic expression of adrenocortical genes (CYP11B1, CYP11B2, CYP21, MC2R) and the Leydig cell specific marker (INSL3) in testicular tissue of fetal 21-hydroxylase deficient (21OHD) mice. Timed-pregnancy studies were performed using H-2aw18 (aw18)-mice. Testes and adrenals of E15.5 and E18.5 mouse fetuses were used for real-time PCR and immunohistochemistry. Gene expression levels were analyzed for genotype-dependent alterations and compared with immunohistochemistry. While enzymes of steroidogenesis showed a significant increased expression in adrenals of 21OHD mice at both E15.5 and E18.5 compared to wild-type (WT) mice, expression levels were unaltered in testes of 21OHD mice. When compared to WT adrenals a significant increase of INSL3 expression in adrenals of 21OHD mice at E15.5 and E18.5 was detected. Cells with adrenocortical properties in mice fetal testis differ from in situ adrenocortical cells in gene expression and growth at E15.5 and E18.5. These findings suggest that the different local regulation and different local niche in adrenals and testes influence growth of aberrant adrenal cells.

Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain.

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.

Selectivity of osilodrostat as an inhibitor of human steroidogenic cytochromes P450.

Osilodrostat (LCI699) is a potent inhibitor of the human steroidogenic cytochromes P450 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). LCI699 is FDA-approved for the treatment of Cushing disease, which is characterized by chronic overproduction of cortisol. While phase II and III clinical studies have proven the clinical efficacy and tolerability of LCI699 for treating Cushing disease, few studies have attempted to fully assess the effects of LCI699 on adrenal steroidogenesis. To this end, we first comprehensively analyzed LCI699-mediated inhibition of steroid synthesis in the NCI-H295R human adrenocortical cancer cell line. We then studied LCI699 inhibition using HEK-293 or V79 cells stably expressing individual human steroidogenic P450 enzymes. Our studies using intact cells confirm the potent inhibition of CYP11B1 and CYP11B2 with negligible inhibition of 17-hydroxylase/17,20-lyase (CYP17A1) and 21-hydroxylase (CYP21A2). Furthermore, partial inhibition of the cholesterol side-chain cleavage enzyme (CYP11A1) was observed. To calculate the dissociation constant (Kd) of LCI699 with the adrenal mitochondrial P450 enzymes, we successfully incorporated P450s into lipid nanodiscs and carried out spectrophotometric equilibrium and competition binding assays. Our binding experiments confirm the high affinity of LCI699 to CYP11B1 and CYP11B2 (Kd ≈ 1 nM or less) and much weaker binding for CYP11A1 (Kd = 18.8 µM). Our results confirm the selectivity of LCI699 for CYP11B1 and CYP11B2 and demonstrate partial inhibition of CYP11A1 but not CYP17A1 and CYP21A2.

cyp21a2 Knockout Tadpoles Survive Metamorphosis Despite Low Corticosterone.

Corticosteroids are so vital for organ maturation that reduced corticosteroid signaling during postembryonic development causes death in terrestrial vertebrates. Indeed, death occurs at metamorphosis in frogs lacking proopiomelanocortin (pomc) or the glucocorticoid receptor (GR; nr3c1). Some residual corticosteroids exist in pomc mutants to activate the wild-type (WT) GR and mineralocorticoid receptor (MR), and the elevated corticosteroids in GR mutants may activate MR. Thus, we expected a more severe developmental phenotype in tadpoles with inactivation of 21-hydroxylase, which should eliminate all interrenal corticosteroid biosynthesis. Using CRISPR/Cas9 in Xenopus tropicalis, we produced an 11-base pair deletion in cyp21a2, the gene encoding 21-hydroxylase. Growth and development were delayed in cyp21a2 mutant tadpoles, but unlike the other frog models, they survived metamorphosis. Consistent with an absence of 21-hydroxylase, mutant tadpoles had a 95% reduction of aldosterone in tail tissue, but they retained some corticosterone (∼40% of WT siblings), an amount, however, too low for survival in pomc mutants. Decreased corticosteroid signaling was evidenced by reduced expression of corticosteroid-response gene, klf9, and by impaired negative feedback in the hypothalamus-pituitary-interrenal axis with higher messenger RNA expression levels of crh, pomc, star, and cyp11b2 and an approximately 30-fold increase in tail content of progesterone. In vitro tail-tip culture showed that progesterone can transactivate the frog GR. The inadequate activation of GR by corticosterone in cyp21a2 mutants was likely compensated for by sufficient corticosteroid signaling from other GR ligands to allow survival through the developmental transition from aquatic to terrestrial life.

A Comprehensive Investigation of Steroidogenic Signaling in Classical and New Experimental Cell Models of Adrenocortical Carcinoma.

Adrenocortical carcinoma is a heterogeneous and aggressive cancer that originates from steroidogenic cells within the adrenal cortex. In this study, we have assessed for the preclinical gold standard NCI-H295 in direct comparison with the more recently established MUC-1 and a here newly reported ACC cell line (TVBF-7) the mutational status of important driver genes (TP53, MEN1, PRKAR1A, CTNNB1, APC, ZNRF-3, IGF-2, EGFR, RB1, BRCA1, BRCA2, RET, GNAS and PTEN), Wnt-signaling specificities (CTNNB1 mutation vs. APC mutation vs. wildtype), steroidogenic-(CYP11A1, CYP17A1, HSD3B2, HSD17B4, CYP21A2, CYP11B1, CYP11B2, MC2R, AT1R) and nuclear-receptor-signaling (AR, ER, GCR), varying electrophysiological potentials as well as highly individual hormone secretion profiles (Cortisol, Aldosterone, DHEA, DHEAS, Testosterone, 17-OH Progesterone, among others) which were investigated under basal and stimulated conditions (ACTH, AngII, FSK). Our findings reveal important genetic and pathophysiological characteristics for these three cell lines and reveal the importance of such cell-line panels reflecting differential endocrine functionalities to thereby better reflect clinically well-known ACC patient heterogeneities in preclinical studies.

Increased Prevalence of Fractures in Congenital Adrenal Hyperplasia: A Swedish Population-based National Cohort Study.

CONTEXT: Low bone mineral density has been reported in individuals with congenital adrenal hyperplasia (CAH), but the prevalence of fractures is unclear. OBJECTIVE: To study the prevalence of fractures in CAH. DESIGN, SETTING, AND PARTICIPANTS: Patients with CAH (n = 714, all 21-hydroxylase deficiency) were compared with controls matched for sex and year and place of birth (n = 71 400). Data were derived by linking National Population-Based Registers. MAIN OUTCOME MEASURES: Number and type of fractures. RESULTS: Mean age was 29.8 ±â€…18.4 years. Individuals with CAH had more fractures compared to controls [23.5% vs 16.1%, odds ratio (OR) 1.61, 95% CI 1.35-1.91], and this was found in both sexes (females: 19.6% vs 13.3%, OR 1.57, 95% CI 1.23-2.02; males: 28.7% vs 19.6%, OR 1.65, 95% CI 1.29-2.12). Fractures were significantly increased in patients born before the introduction of neonatal screening but not in those born afterwards. Any major fracture associated with osteoporosis (spine, forearm, hip, or shoulder) was increased in all individuals with CAH (9.8% vs 7.5%, OR 1.34, 95% CI 1.05-1.72). The highest prevalence of fractures was seen in SV phenotype and I172N genotype while nonclassic phenotype and I2 splice genotype did not show increased prevalence. A transport accident as a car occupant and fall on the same level were more common in patients with CAH, both sexes, than in controls. CONCLUSIONS: Patients with CAH had an increased prevalence of both any fracture and fractures associated with osteoporosis (both sexes) but not for patients neonatally screened. We conclude that fracture risk assessment and glucocorticoid optimization should be performed regularly.

Abordagem da hiperplasia adrenal congênita pela deficiência da enzima 21-hidroxilase em crianças e adolescentes: revisão / Management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in children and adolescents: review

Objetivo: Descrever o diagnóstico e manejo clínico da deficiência da 21-hidroxilase (D-21OH), no contexto atual de inclusão da doença nos programas de triagem neonatal, bem como características genéticas, fisiopatológicas e manifestações na infância e adolescência. Fonte de Dados: Revisão integrativa realizada nas bases de dados MEDLINE (PubMed), LILACS (BVS), Scopus, Web of Science nos últimos vinte anos, em língua inglesa e portuguesa; população-alvo: crianças da primeira infância à adolescência; com o uso dos termos "triagem neonatal", "hiperplasia adrenal congênita", "deficiência da 21-hidroxilase", "glucocorticoide" e "polimorfismos do gene NR3C1". Síntese de Dados: A hiperplasia adrenal congênita (HAC) constitui um grupo de doenças caracterizadas por deficiências enzimáticas na esteroidogênese do córtex adrenal. A D-21OH é responsável por 95% dos casos e, se não tratada precocemente, pode levar ao óbito no período neonatal em sua forma clássica. A triagem neonatal para a HAC consiste na dosagem do precursor 17-hidroxiprogesterona (17OHP) no sangue de recém-nascidos, permitindo rápida confirmação diagnóstica e instituição da terapêutica. A implantação da triagem neonatal constitui um avanço, mas o controle dos pacientes pediátricos com D-21OH é complexo e deve ser sempre individualizado. Conclusão: A instituição dos programas de triagem neonatal para HAC tem trazido benefícios para o prognóstico das crianças com D-21OH. Seu manejo é multiprofissional, individualizado e ainda um desafio mesmo para o especialista. Ampla divulgação do conhecimento sobre a doença é desejável para permitir melhor condução dessas crianças, especialmente de meninas com a doença que apresentam genitália atípica.

Objective: To describe the diagnosis and clinical management of 21-hydroxylase deficiency (21OH-D), in the current context of including the disease in neonatal screening programs, as well as genetic, pathophysiological characteristics, and manifestations in childhood and adolescence. Data Source: Integrative review performed in MEDLINE (PubMed), LILACS (BVS), Scopus, Web of Science databases in the last twenty years, in English and Portuguese; target population: children from early childhood to adolescence; with the use of the terms "neonatal screening"; "congenital adrenal hyperplasia"; "21-hydroxylase deficiency"; "glucocorticoid"; "polymorphisms of the NR3C1 gene". Data Synthesis: Congenital adrenal hyperplasia (CAH) is a group of diseases characterized by enzyme deficiencies in adrenal cortex steroidogenesis. 21OH-D is responsible for 95% of cases and, if not treated early, can lead to death in the neonatal period in its classic form. Neonatal screening for CAH consists of measuring the precursor 17-hydroxyprogesterone (17OHP) in the blood of newborns, allowing rapid diagnostic confirmation and institution of therapy. The implementation of neonatal screening is an advance, but the control of pediatric patients with 21OH-D is complex and must always be individualized. Conclusion: The institution of newborn screening programs for CAH has benefits for the prognosis of children with 21OH-D. Its management is multi-professional, individualized and still a challenge even for the specialist. Wide dissemination of knowledge about the disease is desirable to allow better management of these children, especially girls with the disease who have atypical genitalia.

Corticosteroid Biosynthesis Revisited: No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase.

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.

Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase.

Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.

Challenges of CYP21A2 genotyping in children with 21-hydroxylase deficiency: determination of genotype-phenotype correlation using next generation sequencing in Southeastern Anatolia.

BACKGROUND/PURPOSE: Although it is known that there is generally a good correlation between genotypes and phenotypes, the number of studies reporting discrepancies has recently increased, exclusively between milder genotypes and their phenotypes due to the complex nature of the CYP21A2 gene and methodological pitfalls. This study aimed to assess CYP21A2 genotyping in children with 21-hydroxylase deficiency (21-OHD) and establish their predictive genotype-phenotype correlation features using a large cohort in Southeastern Anatolia's ethnically diverse population. METHODS: The patients were classified into three groups: salt-wasting (SW), simple virilizing (SV) and non-classical (NC). The genotypes were categorized into six groups due to residual enzyme activity: null-A-B-C-D-E. CYP21A2 genotyping was performed by sequence-specific primer and sequenced with next generation sequencing (NGS), and the expected phenotypes were compared to the observed phenotypes. RESULTS: A total of 118 unrelated children with 21-OHD were included in this study (61% SW, 24.5% SV and 14.5% NC). The pathogenic variants were found in 79.5% of 171 mutated alleles (60.2%, 22.2%, and 17.6% in SW, SV and NC, respectively). Patient distribution based on genotype groups was as follows: null-16.1%, A-41.4%, B-6.0%, C-14.4%, E-22%). In2G was the most common pathogenic variant (33.9% of all alleles) and the most common variant in the three phenotype groups (SW-38.8%, SV-22.2% and NC-23.3%). The total genotype-phenotype correlation was 81.5%. The correlations of the null and A groups were 100% and 76.1%, respectively, while it was lower in group B and poor in group C (71.4% and 23.5%, respectively). CONCLUSION: This study revealed that the concordance rates of the severe genotypes with their phenotypes were good, while those of the milder genotypes were poor. The discrepancies could have resulted from the complex characteristics of 21-OHD genotyping and the limitations of using NGS alone without integrating with other comprehensive methods.

Combined homozygous 21 hydroxylase with heterozygous P450 oxidoreductase mutation in a Saudi boy presented with hypertension.

Congenital adrenal hyperplasia (CAH) comprises a group of inherited autosomal recessive disorders characterised by defective cortisol biosynthesis, compensatory increases in corticotrophin secretion and adrenocortical hyperplasia. The characteristics of the biochemical and clinical phenotype depend on the specific enzymatic defect. 21-hydroxylase deficiency is estimated to account for 90%-95% of all CAH cases. Although there are many variants of CAH, a new variant is found secondary to a mutation in the gene encoding the protein P450 oxidoreductase (POR) in which the electron is granted to all microsomal P450 enzymes type II. In 2004, it was discovered that this new CAH disease was attributable to the POR gene mutation. POR facilitates electron transfer from Nicotinamide adenine dinucleotide phosphate (NADPH) to key enzymes involved in steroid and sterol synthesis and metabolism. POR deficiency causes partial and combined impairment of the key enzymes involved in steroidogenesis: P450c17 (17α-hydroxylase/17,20 lyase), P450c21 (21-hydroxylase) and P450aro (aromatase). Clinically, mutant POR manifests with disordered sex development, adrenal insufficiency and skeletal malformations. However, each enzyme may be differently compromised in the same patient. This difference in the clinical manifestations secondary to the variability in enzymatic impairments ranges from ambiguous genitalia in both sexes, adrenal insufficiency associated or not to bone malformations, to abnormal laboratory results in the neonatal screening test of asymptomatic newborns. We report here a case of a 46, XY patient with normal male genitalia associated with hypertension not related to fludrocortisone in which genetic study showed that a homozygous mutation in the CYP21A2 also carries the heterozygous missense variant of unclear pathogenicity in the POR gene.Although there are many variants of CAH, a new variant is found secondary to a mutation in the gene encoding the protein P450 oxidoreductase (POR) which therefore the electron is granted to all microsomal P450 enzymes type II. In 2004, it was mentioned by Fluck and his colleagues that this new CAH disease was attributable to the POR gene mutation.POR facilitates electron transfer from NADPH to key enzymes involved in steroid and sterol synthesis and metabolism.POR deficiency causes partial and combined impairment of the key enzymes involved in steroidogenesis: P450c17 (17α- hydroxylase/17,20 lyase), P450c21 (21-hydroxylase) and P450aro (aromatase).Clinically, Mutant POR manifests with disordered sex development, adrenal insufficiency and skeletal malformations.However, each enzyme may be differently compromised in the same patient. This difference in the clinical manifestations secondary to the variability in enzymatic impairments, it is ranging from ambiguous genitalia in both sexes, adrenal insufficiency associated or not to bone malformations, to abnormal laboratory results in the neonatal screening test of asymptomatic newborns.

Non-Classic Disorder of Adrenal Steroidogenesis and Clinical Dilemmas in 21-Hydroxylase Deficiency Combined with Backdoor Androgen Pathway. Mini-Review and Case Report.

Congenital adrenal hyperplasia (CAH) is the most common cause of primary adrenal insufficiency in children and adolescents. It comprises several clinical entities associated with mutations in genes, encoding enzymes involved in cortisol biosynthesis. The mutations lead to considerable (non-classic form) to almost complete (classic form) inhibition of enzymatic activity, reflected by different phenotypes and relevant biochemical alterations. Up to 95% cases of CAH are due to mutations in CYP21A2 gene and subsequent 21α-hydroxylase deficiency, characterized by impaired cortisol synthesis and adrenal androgen excess. In the past two decades an alternative ("backdoor") pathway of androgens' synthesis in which 5α-androstanediol, a precursor of the 5α-dihydrotestosterone, is produced from 17α-hydroxyprogesterone, with intermediate products 3α,5α-17OHP and androsterone, in the sequence and with roundabout of testosterone as an intermediate, was reported in some studies. This pathway is not always considered in the clinical assessment of patients with hyperandrogenism. The article describes the case of a 17-year-old female patient with menstrual disorders and androgenization (persistent acne, advanced hirsutism). Her serum dehydroepiandrosterone sulfate and testosterone were only slightly elevated, along with particularly high values for 5α-dihydrotestosterone. In 24 h urine collection, an increased excretion of 16α-OHDHEA-a dehydroepiandrosterone metabolite-and pregnanetriolone-a 17α-hydroxyprogesterone metabolite-were observed. The investigations that we undertook provided evidence that the girl suffered from non-classic 21α-hydroxylase deficiency with consequent enhancement of the androgen "backdoor" pathway in adrenals, peripheral tissues or both, using adrenal origin precursors. The paper presents diagnostic dilemmas and strategies to differentiate between various reasons for female hyperandrogenism, especially in childhood and adolescence.

How do mutations affect the structural characteristics and substrate binding of CYP21A2? An investigation by molecular dynamics simulations.

Congenital adrenal hyperplasia (CAH) is one of the most frequent inborn errors of metabolism, inherited as an autosomal recessive trait. Above 95% of CAH cases are caused by mutations in cytochrome P450 21A2 (CYP21A2). It is a pity that how these mutations affect the structural characteristics and substrate binding of CYP21A2 is still unclear. To this end, molecular dynamics (MD) simulations and binding free energy calculations are performed to investigate the effects of single point mutations (L108R, G292C, G292S, G293D, and T296N) in CYP21A2. The results indicate that mutations could cause the local conformational changes of CYP21A2, affecting the substrate binding by changing the interaction between the protein and heme, changing the charge environment of residues, or introducing steric hindrance. In addition, our work gives a wonderful explanation of the phenomenon that though the substrate binding ability increases, the reaction activity decreases in T296N. The present study provides detailed atomistic insights into the structure-function relationships of CYP21A2, which could contribute to further understanding about 21-hydroxylase deficiency and also provide a theoretical basis for CAH prediction and treatment.

Mixed-culture fermentation for enhanced C21-hydroxylation of glucocorticoids.

Synthetic glucocorticoids are generally preferred over their natural counterparts as these compounds exhibit improved anti-inflammatory potency and glucocorticoid receptor selectivity. However, the biotechnological production of these molecules is often subject to limitations inferred by restricted enzyme stability, selectivity or inhibition thereof. The latter is particularly important during 6α-methylprednisolone production, as the essential C21-hydroxylation of its precursor medrane appears to be hampered by product inhibition of the steroid-21-hydroxylase (CYP21A2). To circumvent this bottleneck, we established a two-step reaction for controlled mixed-culture fermentation, using recombinant E. coli. This process comprises the previously reported C21-hydroxylation of medrane by CYP21A2, followed by an instant derivatization of the hydroxylated product premedrol by chloramphenicol acetyl transferase 1 (CAT1). The CAT1-mediated C21-acetylation prevents the product from regaining access to the enzyme's active site which effectively shifts the chemical equilibrium toward premedrol formation. The successful circumvention of product inhibition at optimized conditions resulted in the formation of more than 1.5 g of product per liter which corresponds to an increase by more than 100 %. Taken together, we demonstrate an efficient system to enhance cytochrome P450-mediated biotransformations, holding great ecologic and economic potential to be applied in industrial processes.

A rare CYP21A2 haplotype clarifies the phenotype-genotype discrepancy in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH).

RCCX haplotypes with two copies of the CYP21A2 gene and one copy of the CYP21A1P pseudogene have been widely described in different populations. In most cases, the CYP21A2-like gene downstream of the TNXA gene showed a wild-type sequence or the c.293-13A/C > G variant while the CYP21A2 gene next to TNXB carried the p.(Gln319Ter) variant. Here is the discovery of a novel rare CYP21A2 haplotypes detected in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH). The molecular family study was performed clarifying the previously found phenotype-genotype discrepancy.

Pregnancy-associated changes of peroxisome proliferator-activated receptor delta (PPARD) and cytochrome P450 family 21 subfamily A member 2 (CYP21A2) expression in the bovine corpus luteum.

We investigated gene expression profiles of the corpus luteum (CL) at the time of maternal recognition to evaluate the functional changes of the CL during early pregnancy in cows and help improve reproductive efficiency and avoid defective fetuses. Microarray analyses using a 15 K bovine oligo DNA microarray detected 30 differentially expressed genes and 266 differentially expressed genes (e.g., PPARD and CYP21A2) in the CL on pregnancy days 15 (P15) and 18 (P18), respectively, compared with the CL on day 15 (NP15) of non-pregnancy (n = 4 for each group). PPARD expression was the highest while the CYP21A2 expression was the lowest in P15 and P18 compared with that of NP15. These microarray results were validated by quantitative real-time PCR analysis. The addition of interferon-τ and supernatants derived from homogenized fetal trophoblast increased ISG15 and MX1 expressions in the cultured luteal tissue (P < 0.01), but did not affect PPARD and CYP21A2 expressions. PPARD expression in the luteal tissue was stimulated (P < 0.05) by GW0742, known as a selective PPARD agonist, and PPARD ligands (i.e., arachidonic, linoleic and linolenic acids). In contrast, CYP21A2 mRNA expression was not affected by both agonist and ligands. The concentration of prostaglandin (PG) E2 and PGF2α decreased after GW0742 stimulation and increased after arachidonic acid stimulation (P < 0.05). The addition of GW0742 and arachidonic acid increased progesterone (P4) concentration. Collectively, these findings suggest that high expression levels of PPARD and low expression levels of CYP21A2 in the CL during early pregnancy may support P4 production by bovine luteal cells.

Estimation of the inhibiting impact of abiraterone D4A metabolite on human steroid 21-monooxygenase (CYP21A2).

Abiraterone D4A metabolite, the product of 3ß-hydroxysteroid dehydrogenase activity toward abiraterone, may serve as a potential antitumor agent for the treatment of prostate cancer. The main adverse effect of abiraterone is the disruption of corticosteroid biosynthesis, and the more pharmacologically active abiraterone D4A metabolite may have the same issues. We therefore estimated the inhibiting impact of the abiraterone D4A metabolite on one of the key corticosteroidogenic enzymes - human steroid 21-monooxygenase (CYP21A2). Molecular docking of D4A into the active site of CYP21A2 has been predicted to be similar to abiraterone binding with the enzyme. Abiraterone D4A metabolite, similar to abiraterone, induces type II spectral changes of CYP21A2. The spectral dissociation constant for the abiraterone D4A metabolite-CYP21A2 complex was calculated as 3.4 ±â€¯0.5 µM. Abiraterone D4A metabolite demonstrates competitive/mixed type CYP21A2 inhibition with an inhibitory constant of 1.8 ±â€¯0.8 µM, as obtained by Dixon plot. These results make it possible to predict the adverse effects of the new perspective candidate compound for antitumor therapy.

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.

Expression of genes encoding mineralocorticoid biosynthetic enzymes and the mineralocorticoid receptor, and levels of mineralocorticoids in the bovine follicle and corpus luteum.

Unlike sex steroids, mineralocorticoids have attracted limited attention in ovarian physiology. Recent studies on primates have indicated possible local synthesis and action of mineralocorticoids in the ovary. Here, we examined developmental changes in the levels of mineralocorticoids and expression of genes encoding their biosynthetic enzymes and receptor in the bovine ovary. The follicles and corpora lutea (CL) were collected from F1 heifers. Expression levels of 21α-hydroxylase (CYP21A2), 11ß-hydroxylase-1 (CYP11B1), and the mineralocorticoid receptor (NR3C2) in granulosa cells (GC), thecal layers (TL), and CL tissues were quantified by real-time PCR, whereas mineralocorticoids in the follicular fluid were measured by enzyme immunoassay (EIA). TL and GC expressed CYP21A2 and NR3C2, whereas CYP11B1 was expressed at very low or undetectable levels. The expression levels of these genes were not significantly different among small/large and healthy/atretic follicles but were higher in TL than in GC. CYP21A2 and NR3C2 were expressed in all CL stages with higher expression observed in the mid-stage. CYP11B1 expression was only apparent in the mid-stage CL. Aldosterone was detected in all follicles, and its concentration was not significantly different among the follicular groups. In paired large-healthy/atretic follicles, the concentration of deoxycorticosterone, a precursor of aldosterone, was approximately ten-fold higher than that of aldosterone and not significantly different between healthy and atretic follicles. In conclusion, the presence of mineralocorticoids and expression of NR3C2 in the bovine follicle together with the developmental change in the expression of CYP21A2, CYP11B1, and NR3C2 in the CL suggest possible endocrine/paracrine/autocrine roles of mineralocorticoids in the bovine ovary.