Characterization of human adrenal cytochrome P450 11B2 products of progesterone and androstenedione oxidation

The Journal of steroid biochemistry and molecular biology

Published On 2021/4/1

Cytochrome P450 (P450) 11B1 and 11B2 both catalyze the 11β-hydroxylation of 11-deoxycorticosterone and the subsequent 18-hydroxylation of the product. P450 11B2, but not P450 11B1, catalyzes a further C-18 oxidation to yield aldosterone. 11-Oxygenated androgens are of interest, and 11-hydroxy progesterone has been reported to be a precursor of these. Oxidation of progesterone by purified recombinant P450 11B2 yielded a mono-hydroxy derivative as the major product, and co-chromatography with commercial standards and 2-D NMR spectroscopy indicated 11β-hydroxylation. 18-Hydroxyprogesterone and a dihydroxyprogesterone were also formed. Similarly, oxidation of androstenedione by P450 11B2 yielded 11β-hydroxyandrostenedione, 18-hydroxyandrostenedione, and a dihydroxyandrostenedione. The steady-state kinetic parameters for androstenedione and progesterone 11β-hydroxylation …

Journal

The Journal of steroid biochemistry and molecular biology

Published On

2021/4/1

Volume

208

Page

105787

Authors

Fred Guengerich

Fred Guengerich

Vanderbilt University

Position

Professor of Biochemistry

H-Index(all)

175

H-Index(since 2020)

51

I-10 Index(all)

0

I-10 Index(since 2020)

0

Citation(all)

0

Citation(since 2020)

0

Cited By

0

Research Interests

Enzymology

drug metabolism

cytochrome P450

mutagenesis

University Profile Page

Michael Reddish

Michael Reddish

Appalachian State University

Position

H-Index(all)

12

H-Index(since 2020)

9

I-10 Index(all)

0

I-10 Index(since 2020)

0

Citation(all)

0

Citation(since 2020)

0

Cited By

0

Research Interests

Biochemistry

Enzymology

Spectroscopy

University Profile Page

Stella Child

Stella Child

Vanderbilt University

Position

H-Index(all)

7

H-Index(since 2020)

7

I-10 Index(all)

0

I-10 Index(since 2020)

0

Citation(all)

0

Citation(since 2020)

0

Cited By

0

Research Interests

Cytochrome P450

University Profile Page

Sarah M Glass

Sarah M Glass

Vanderbilt University

Position

Graduate Student

H-Index(all)

6

H-Index(since 2020)

6

I-10 Index(all)

0

I-10 Index(since 2020)

0

Citation(all)

0

Citation(since 2020)

0

Cited By

0

Research Interests

Drug Metabolism

Biochemistry

University Profile Page

Other Articles from authors

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Medicinal Chemistry

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Chemical Research in Toxicology

In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 μM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Formation of potentially toxic metabolites of drugs in reactions catalyzed by human drug-metabolizing enzymes

Data are presented on the formation of potentially toxic metabolites of drugs that are substrates of human drug metabolizing enzymes. The tabular data lists the formation of potentially toxic/reactive products. The data were obtained from in vitro experiments and showed that the oxidative reactions predominate (with 96% of the total potential toxication reactions). Reductive reactions (e.g., reduction of nitro to amino group and reductive dehalogenation) participate to the extent of 4%. Of the enzymes, cytochrome P450 (P450, CYP) enzymes catalyzed 72% of the reactions, myeloperoxidase (MPO) 7%, flavin-containing monooxygenase (FMO) 3%, aldehyde oxidase (AOX) 4%, sulfotransferase (SULT) 5%, and a group of minor participating enzymes to the extent of 9%. Within the P450 Superfamily, P450 Subfamily 3A (P450 3A4 and 3A5) participates to the extent of 27% and the Subfamily 2C (P450 2C9 and P450 2C19 …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Proteomics, modeling, and fluorescence assays delineate cytochrome b5 residues involved in binding and stimulation of cytochrome P450 17A1 17, 20-lyase

Cytochrome b5 (b5) is known to stimulate some catalytic activities of cytochrome P450 (P450, CYP) enzymes, although mechanisms still need to be defined. The reactions most strongly enhanced by b5 are the 17,20-lyase reactions of P450 17A1 involved in steroid biosynthesis. We had previously used a fluorescently labeled human b5 variant (Alexa 488-T70C-b5) to characterize human P450 17A1-b5 interactions, but subsequent proteomic analyses indicated that lysines in b5 were also modified with Alexa 488 maleimide in addition to Cys-70, due to disulfide dimerization of the T70C mutant. A series of b5 variants were constructed with Cys replacements for the identified lysine residues and labeled with the dye. Fluorescence attenuation and the function of b5 in the steroid lyase reaction depended on the modified position. Apo-b5 (devoid of heme group) studies revealed the lack of involvement of the b5 heme in …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Angewandte Chemie

Oxygen‐18 Labeling Reveals a Mixed Fe− O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α‐Demethylation

The 14α‐demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three‐step reaction terminates in a C−C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2−) or Compound I (perferryl oxygen, FeO3+) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18O incorporation into formic acid from 18O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18O (one atom) into formic acid, consistent with a major but not exclusive FeO2− mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Ninety-eight semesters of cytochrome P450 enzymes and related topics—What have I taught and learned?

This Reflection article begins with my family background and traces my career through elementary and high school, followed by time at the University of Illinois, Vanderbilt University, the University of Michigan, and then for 98 semesters as a Vanderbilt University faculty member. My research career has dealt with aspects of cytochrome P450 enzymes, and the basic biochemistry has had applications in fields as diverse as drug metabolism, toxicology, medicinal chemistry, pharmacogenetics, biological engineering, and bioremediation. I am grateful for the opportunity to work with the Journal of Biological Chemistry not only as an author but also for 34 years as an Editorial Board Member, Associate Editor, Deputy Editor, and interim Editor-in-Chief. Thanks are extended to my family and my mentors, particularly Profs. Harry Broquist and Minor J. Coon, and the more than 170 people who have trained with me. I have …

Fred Guengerich

Fred Guengerich

Vanderbilt University

ACS catalysis

Oxygen-18 Labeling Defines a Ferric Peroxide (Compound 0) Mechanism in the Oxidative Deformylation of Aldehydes by Cytochrome P450 2B4

Most cytochrome P450 (P450) oxidations are considered to occur with the active oxidant being a perferryl oxygen (FeO3+, Compound I). However, a ferric peroxide (FeO2̅, Compound 0) mechanism has been proposed, as well, particularly for aldehyde substrates. We investigated three of these systems, the oxidative deformylation of the model substrates citronellal, 2-phenylpropionaldehyde, and 2-methyl-2-phenylpropionaldehyde by rabbit P450 2B4, using 18O labeling. The formic acid product contained one 18O derived from 18O2, which is indicative of a dominant Compound 0 mechanism. The formic acid also contained only one 18O derived from H218O, which ruled out a Compound I mechanism. The possibility of a Baeyer–Villiger reaction was examined by using synthesized possible intermediates, but our data do not support its presence. Overall, these findings unambiguously demonstrate the role of the …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Principles of Xenobiotic Metabolism (Biotransformation)

This chapter provides a general overview of metabolic reactions and their significance. Basic concepts and terminology related to biotransformation, activity, and toxicityToxicity are explained and discussed. Major enzymes involved in oxidationOxidation, reductionReduction, hydrolytic, and conjugationConjugation are covered including enzyme nomenclature, localization, catalytic cycle, coenzymes, relevance of individual enzymes, types of reactions, substrates and metabolites, influence of metabolic reactions on the activity/toxicity of xenobiotics, enzyme inhibition, and relevance if applicable.

Stella Child

Stella Child

Vanderbilt University

Archives of Biochemistry and Biophysics

Structural determination and characterisation of the CYP105Q4 cytochrome P450 enzyme from Mycobacterium marinum

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyze important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV–vis absorbance spectrum. Based on the UV–vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate …

2023/11/24

Article Details
Stella Child

Stella Child

Vanderbilt University

Journal of Inorganic Biochemistry

Selective carbon-hydrogen bond hydroxylation using an engineered cytochrome P450 peroxygenase

The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this enzyme into a H2O2 utilizing peroxygenase. This variant displayed significantly increased peroxide driven hydroxylation activity towards the saturated linear fatty acids tested (undecanoic through to hexadecenoic acid) when compared to the wild-type heme domain. The product distributions arising from fatty acid oxidation using this peroxygenase variant were broadly similar to those obtained with the wild-type monooxygenase holoenzyme, with oxidation occurring predominantly at the ω-1 through to ω-3 positions. 10-Undecenoic acid was regioselectively hydroxylated at the allylic ω-2 carbon by the Thr268Glu peroxygenase. The effect of isotopic …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of Biological Chemistry

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450 …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Nucleic Acids Research

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIβ and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Xenobiotica

The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyse warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C.2. Compared with that seen at 37 °C, the intrinsic clearance rates (Vmax/Km) of CYP2C9.1 and .2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1, .2, and .3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and .10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and .23 were increased (130 ∼ 134%). At 40 …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Food and Chemical Toxicology

FEMA GRAS assessment of natural flavor complexes: Lemongrass oil, chamomile oils, citronella oil and related flavoring ingredients

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, eleventh in the series, evaluates the safety of NFCs characterized by primary alcohol, aldehyde, carboxylic acid, ester and lactone constituents derived from terpenoid biosynthetic pathways and/or lipid metabolism. The scientific-based evaluation procedure published in 2005 and updated in 2018 that relies on a complete constituent characterization of the NFC and organization of the constituents into congeneric groups. The safety of the NFCs is evaluated using the threshold of toxicological concern (TTC) concept in addition to data on estimated intake, metabolism and toxicology of members of the congeneric groups and for the NFC under evaluation. The scope of the safety evaluation …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Food and Chemical Toxicology

FEMA GRAS assessment of derivatives of basil, nutmeg, parsley, tarragon and related allylalkoxybenzene-containing natural flavor complexes

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients in food. In this publication, tenth in the series, NFCs containing a high percentage of at least one naturally occurring allylalkoxybenzene constituent with a suspected concern for genotoxicity and/or carcinogenicity are evaluated. In a related paper, ninth in the series, NFCs containing anethole and/or eugenol and relatively low percentages of these allylalkoxybenzenes are evaluated. The Panel applies the threshold of toxicological concern (TTC) concept and evaluates relevant toxicology data on the NFCs and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s), the estimated intake of the constituent is compared to the TTC for compounds with structural …

Fred Guengerich

Fred Guengerich

Vanderbilt University

International Journal of Molecular Sciences

Identification of Three Human POLH Germline Variants Defective in Complementing the UV-and Cisplatin-Sensitivity of POLH-Deficient Cells

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1—432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3’-T and 5′-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants—substantially reduced in TLS activity—failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may …

Fred Guengerich

Fred Guengerich

Vanderbilt University

Journal of inorganic biochemistry

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1)

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance …

Fred Guengerich

Fred Guengerich

Vanderbilt University

The Importance of Biotransformation

Biotransformation is important in considerations of toxicity of chemicals. What begins as a well-defined compound may lead to a mixture of chemicals after it enters the body. The changes may be beneficial or detrimental. A potentially harmful chemical may be rapidly inactivated, at low doses. Conversely, an innocuous compound may be transformed into a toxic one. There are cases of both detoxication and bioactivation for the same chemical, sometimes even with the same enzyme being involved in both changes (e.g., aflatoxin B1 and cytochrome P450 3A4). A proper understanding of the chemical changes, the enzymes involved, and the kinetics of changes is needed to understand the outcomes regarding safety assessment.

Fred Guengerich

Fred Guengerich

Vanderbilt University

Drug Metabolism and Disposition

Cytochrome P450 enzymes as drug targets in human disease

Although the mention of cytochrome P450 (P450, CYP) inhibition usually brings to mind unwanted variability in pharmacokinetics, in several cases P450s are good targets for inhibition. These P450s are essential but in certain disease states it is desirable to reduce the concentrations of their products. Most of the attention to date has been with human P450s 5A1, 11A1, 11B1, 11B2, 17A1, 19A1, and 51A1. In some of those cases, there are multiple drugs in us, e.g., exemestane, letrozole, and anastrozole with P450 19A1, the steroid aromatase target in breast cancer. There are also several targets that are less developed, e. g. P450s 2A6, 8B1, 4A11, 24A1, 26A1, and 26B1.Significance Statement The selective inhibition of certain P450s that have major physiological functions has been shown to be very efficacious in certain human diseases. In several cases the search for better drugs continues.

Other articles from The Journal of steroid biochemistry and molecular biology journal

Katharina Lang

Katharina Lang

University of Birmingham

The Journal of Steroid Biochemistry and Molecular Biology

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5–10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and …

Sandesh Parajuli

Sandesh Parajuli

University of Wisconsin-Madison

The Journal of steroid biochemistry and molecular biology

Association of Circulating 25-Hydroxyvitamin D and Recurrence of Glomerulonephritis in Kidney Transplant Recipients: The Wisconsin Allograft Recipient Database (WisARD)

Evidence on the association of vitamin D and parathyroid hormone (PTH) with cardiovascular risk factors in the young is limited. We therefore assessed the relationships of circulating vitamin D and PTH concentrations and subclinical atherosclerosis in overweight or obese children and adolescents. This was a cross-sectional study, investigated the association of 25-hydroxyvitamin D (25(OH)D), intact PTH (iPTH), and iPTH/25(OH)D ratio with carotid intima–media thickness (cIMT) in 368 Iranian children and adolescents with a body mass index (BMI) ≥1 z-score based on WHO criteria. Ultrasound measurement of cIMT was performed. Multivariable linear and logistic regressions were used to test associations between 25(OH)D, iPTH, and iPTH/25(OH)D ratio using one-ln-unit increment with cIMT. Median (25–75 interquartile range) 25(OH)D and iPTH concentrations were 11.8 (8.2–18.6) ng/ml and 38.2 (25.0–61.4 …

Arnout Voet

Arnout Voet

Katholieke Universiteit Leuven

The Journal of Steroid Biochemistry and Molecular Biology

Structural mechanism underlying variations in DNA binding by the androgen receptor

The androgen receptor (AR) is a steroid activated transcription factor which recognizes DNA motifs resembling inverted repeats of a conserved 5’-AGAACA-3’-like hexanucleotides separated by a three-nucleotide spacer from a similar, but less conserved hexanucleotide. Here, we report the structures of the human AR DNA binding domain (DBD) bound to two natural AREs (C3 and MTV) in head-to-head dimer conformations, diffracting at 2.05 Å and 2.25 Å, respectively. These structures help to explain the impact of androgen insensitivity mutations on the structure integrity, DNA binding and DBD dimerization. The binding affinity of the AR DBD to different DNA motifs were measured by the BioLayer Interferometry (BLI) and further validated by Molecular Dynamics (MD) simulations. This shows that the high binding affinity of the first DBD to the upstream 5’-AGAACA-3’ motif induces the cooperative binding of the …

Jaap Deinum

Jaap Deinum

Radboud Universiteit

The Journal of Steroid Biochemistry and Molecular Biology

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5–10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and …

HIRONOBU SASANO

HIRONOBU SASANO

Tohoku University

The Journal of Steroid Biochemistry and Molecular Biology

Progesterone receptor-Grb2 interaction is associated with better outcomes in breast cancer

In addition to mediating nuclear transcription, PR mediates extranuclear functions mainly through the PR polyproline domain (PPD) interaction with the SH3 domain of cytoplasmic signaling molecules. PR-PPD-SH3 interaction inhibits EGF-mediated signaling and decreases lung cancer cell proliferation. Grb2 is an essential adaptor molecule with an SH2 domain flanked by two SH3 domains. In this study, we examined whether PR, through interaction between PR-PPD and Grb2-SH3, can interact with Grb2 in cells and breast cancer tissues. Our previous study shows that interaction between PR-PPD and Grb2 could interfere with cytoplasmic signaling and lead to inhibition of EGF-mediated signaling. GST-pulldown analysis shows that PR-PPD specifically interacts with the SH3 domains of Grb2. Immunofluorescence staining shows colocalization of PR and Grb2 in both the nucleus and cytoplasm in BT-474 breast …

Frank Claessens

Frank Claessens

Katholieke Universiteit Leuven

The Journal of Steroid Biochemistry and Molecular Biology

Structural mechanism underlying variations in DNA binding by the androgen receptor

The androgen receptor (AR) is a steroid activated transcription factor which recognizes DNA motifs resembling inverted repeats of a conserved 5’-AGAACA-3’-like hexanucleotides separated by a three-nucleotide spacer from a similar, but less conserved hexanucleotide. Here, we report the structures of the human AR DNA binding domain (DBD) bound to two natural AREs (C3 and MTV) in head-to-head dimer conformations, diffracting at 2.05 Å and 2.25 Å, respectively. These structures help to explain the impact of androgen insensitivity mutations on the structure integrity, DNA binding and DBD dimerization. The binding affinity of the AR DBD to different DNA motifs were measured by the BioLayer Interferometry (BLI) and further validated by Molecular Dynamics (MD) simulations. This shows that the high binding affinity of the first DBD to the upstream 5’-AGAACA-3’ motif induces the cooperative binding of the …

Luigi Iuliano

Luigi Iuliano

Sapienza Università di Roma

The Journal of Steroid Biochemistry and Molecular Biology

11th ENOR meeting: Oxysterols in human health and diseases.

11th ENOR meeting: Oxysterols in human health and diseases. - Abstract - Europe PMC Sign in | Create an account https://orcid.org Europe PMC Menu About Tools Developers Help Contact us Helpdesk Feedback Twitter Blog Tech blog Developer Forum Europe PMC plus Search life-sciences literature (43,705,148 articles, preprints and more) Search Advanced search Feedback This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy. Abstract Full text Citations & impact 11 th ENOR meeting: Oxysterols in human health and diseases. Poirot M 1 , Iuliano L 2 , Griffiths WJ 2 , Lizard G 2 Author information Affiliations 1. Cancer Research Center of Toulouse (CRCT), Team "Cholesterol Metabolism and Therapeutic Innovations"; The French Network for Nutrition and Cancer Research (…

Hee-Seok Lee

Hee-Seok Lee

Chung-Ang University

The Journal of Steroid Biochemistry and Molecular Biology

Beauvericin, produced by Fusarium oxysporum inhibits bisphenol A-induced proliferation of human breast cancer cell line by regulating ERα/p38 pathway

Beauvericin (BEA) is a cyclic depsipeptide secondary metabolite of Fusarium species. It causes chemical hazards in food products and exists in an environment containing soil and various food types. On the other hand, the purified BEA has various biological activities and is regarded as a potential candidate for pharmaceutical research. This study was performed to assess the anti-proliferation activity of BEA against human breast cancer cells by regulating the estrogen receptor-alpha (ERα)/p38 pathway. TA and BA assays verified that BEA is a completed ER antagonist. Additionally, BEA suppressed cell proliferation in the anti-proliferation assay involving ER-positive human breast cancer cells co-treated with BPA and BEA. In respect to an anti-proliferation activity, the BPA-induced phosphorylation of p38 protein was inhibited in the presence of BEA. These results suggested that BEA exerts inhibitory potentials on …

William J Griffiths

William J Griffiths

Swansea University

The Journal of Steroid Biochemistry and Molecular Biology

11th ENOR meeting: Oxysterols in human health and diseases.

11th ENOR meeting: Oxysterols in human health and diseases. - Abstract - Europe PMC Sign in | Create an account https://orcid.org Europe PMC Menu About Tools Developers Help Contact us Helpdesk Feedback Twitter Blog Tech blog Developer Forum Europe PMC plus Search life-sciences literature (43,705,148 articles, preprints and more) Search Advanced search Feedback This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy. Abstract Full text Citations & impact 11 th ENOR meeting: Oxysterols in human health and diseases. Poirot M 1 , Iuliano L 2 , Griffiths WJ 2 , Lizard G 2 Author information Affiliations 1. Cancer Research Center of Toulouse (CRCT), Team "Cholesterol Metabolism and Therapeutic Innovations"; The French Network for Nutrition and Cancer Research (…

Viroj Boonyaratanakornkit

Viroj Boonyaratanakornkit

Chulalongkorn University

The Journal of Steroid Biochemistry and Molecular Biology

Progesterone receptor-Grb2 interaction is associated with better outcomes in breast cancer

In addition to mediating nuclear transcription, PR mediates extranuclear functions mainly through the PR polyproline domain (PPD) interaction with the SH3 domain of cytoplasmic signaling molecules. PR-PPD-SH3 interaction inhibits EGF-mediated signaling and decreases lung cancer cell proliferation. Grb2 is an essential adaptor molecule with an SH2 domain flanked by two SH3 domains. In this study, we examined whether PR, through interaction between PR-PPD and Grb2-SH3, can interact with Grb2 in cells and breast cancer tissues. Our previous study shows that interaction between PR-PPD and Grb2 could interfere with cytoplasmic signaling and lead to inhibition of EGF-mediated signaling. GST-pulldown analysis shows that PR-PPD specifically interacts with the SH3 domains of Grb2. Immunofluorescence staining shows colocalization of PR and Grb2 in both the nucleus and cytoplasm in BT-474 breast …

Marcus E Kleber

Marcus E Kleber

Ruprecht-Karls-Universität Heidelberg

The Journal of Steroid Biochemistry and Molecular Biology

Determinants of circulating calcitriol in cardiovascular disease

Circulating calcitriol may contribute to the risk of cardiovascular disease (CVD), but its regulation in patients with CVD is poorly characterized. We therefore aimed to assess determinants of circulating calcitriol in these patients. We analyzed 2183 independent samples from a large cohort of patients scheduled for coronary angiography and 1727 independent samples from different other cohorts from patients with a wide range of CVDs, including heart transplant candidates, to quantify the association of different parameters with circulating calcitriol. We performed univariable and multivariable linear regression analyses using the mathematical function that fitted best with circulating calcitriol. In the multivariable analysis of the large single cohort, nine parameters remained significant, explaining 30.0% (32.4% after exclusion of 22 potential outliers) of the variation in circulating calcitriol (r=0.548). Log-transformed 25 …

Cyrus Jalili

Cyrus Jalili

Kermanshah University of Medical Sciences

The Journal of Steroid Biochemistry and Molecular Biology

The influence of indole propionic acid on molecular markers of steroidogenesis, ER stress, and apoptosis in rat granulosa cells exposed to high glucose conditions

Hyperglycemia is known as one of the main causes of infertility in human societies. Indole propionic acid (IPA) is produced by intestinal microbiota and has antioxidant and anti-inflammatory properties. This study aims to investigate the effects of IPA on molecular indices of steroidogenesis, ER stress, and apoptosis induced by high glucose (HG) in granulosa cells. Primary GCs, isolated from ovarian follicles of Rats were cultured in 5 mM (control) and 30 mM (HG) of glucose and in the presence of 10 and 20 µM of IPA for 24 h. The cell viability was assessed by MTT. The gene expression of P450SCC, 3βHSD, CYP19A, BAX, BCL2, and STAR was evaluated by Real-Time PCR. Protein expression of ATF6, PERK, GRP78, and CHOP determined by western blot. Progesterone, estradiol, IL-1β, and TNF-α were measured by ELISA. HG decreased the viability, and expression of P450SCC, 3βHSD, CYP19A, BCL2 …

Alice J Sitch

Alice J Sitch

University of Birmingham

The Journal of Steroid Biochemistry and Molecular Biology

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5–10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and …

Brad Astor

Brad Astor

University of Wisconsin-Madison

The Journal of steroid biochemistry and molecular biology

Association of Circulating 25-Hydroxyvitamin D and Recurrence of Glomerulonephritis in Kidney Transplant Recipients: The Wisconsin Allograft Recipient Database (WisARD)

Evidence on the association of vitamin D and parathyroid hormone (PTH) with cardiovascular risk factors in the young is limited. We therefore assessed the relationships of circulating vitamin D and PTH concentrations and subclinical atherosclerosis in overweight or obese children and adolescents. This was a cross-sectional study, investigated the association of 25-hydroxyvitamin D (25(OH)D), intact PTH (iPTH), and iPTH/25(OH)D ratio with carotid intima–media thickness (cIMT) in 368 Iranian children and adolescents with a body mass index (BMI) ≥1 z-score based on WHO criteria. Ultrasound measurement of cIMT was performed. Multivariable linear and logistic regressions were used to test associations between 25(OH)D, iPTH, and iPTH/25(OH)D ratio using one-ln-unit increment with cIMT. Median (25–75 interquartile range) 25(OH)D and iPTH concentrations were 11.8 (8.2–18.6) ng/ml and 38.2 (25.0–61.4 …

jin-song shi

jin-song shi

Jiangnan University

The Journal of Steroid Biochemistry and Molecular Biology

Engineering of redox partners and cofactor NADPH supply of CYP68JX for efficient steroid two-step ordered selective hydroxylation activity

CYP68JX, a P450 hydroxylase, derived from Colletotrichum lini ST-1 is capable of biotransforming dehydroepiandrosterone (DHEA) to 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA). Redox partners and cofactor supply are important factors affecting the catalytic activity of CYP68JX. In this study, the heterologous expression of CYP68JX in Saccharomyces cerevisiae BY4741 was realized resulting in a 17.1% target product yield. In order to increase the catalytic efficiency of CYP68JX in S. cerevisiae BY4741, a complete cytochrome P450 redox system was constructed. Through the combination of CYP68JX and heterologous CPRs, the yield of the target product 7α,15α-diOH-DHEA in CYP68JX recombinant system was increased to 37.8%. Furthermore, by adding NADPH coenzyme precursor tryptophan of 40 mmol/L and co-substrate fructose of 20 g/L during the conversion process, the catalytic …

Knigge Thomas

Knigge Thomas

Université du Havre

The Journal of Steroid Biochemistry and Molecular Biology

Evolutionary history and functional characterization of duplicated G protein-coupled estrogen receptors in European sea bass

Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish …

HUMBERTO L. MENDOZA FIGUEROA

HUMBERTO L. MENDOZA FIGUEROA

Instituto Politécnico Nacional

The Journal of Steroid Biochemistry and Molecular Biology

GPER binding site detection and description: A flavonoid-based docking and molecular dynamics simulations study.

Flavonoids, a phenolic compounds class widely distributed in the plant kingdom, have attracted much interest for their implications on several health and disease processes. Usually, the consumption of this type of compounds is approximately 1 g/d, primarily obtained from cereals, chocolate, and dry legumes ensuring its beneficial role in maintaining the homeostasis of the human body. In this context, in cancer disease prominent data points to the role of flavonoids as adjuvant treatment aimed at the regression of the disease. GPER, an estrogen receptor on the cell surface, has been postulated as a probable orchestrator of the beneficial effects of several flavonoids through modulation/inhibition of various mechanisms that lead to cancer progression. Therefore, applying pocket and cavity protein detection and docking and molecular dynamics simulations (MD), we generate, from a cluster composed of 39 flavonoids …

Carolina Meloni Vicente

Carolina Meloni Vicente

Universidade Federal de São Paulo

The Journal of Steroid Biochemistry and Molecular Biology

Activation of estrogen receptor induces differential proteomic responses mainly involving migration, invasion, and tumor development pathways in human testicular embryonal …

The aims of the present study were to investigate the global changes on proteome of human testicular embryonal carcinoma NT2/D1 cells treated with 17β-estradiol (E2), and the effects of this hormone on migration, invasion, and colony formation of these cells. A quantitative proteomic analysis identified the presence of 1230 proteins in both E2-treated and control cells. The analysis revealed 75 differentially abundant proteins (DAPs), out of which 43 proteins displayed a higher abundance and, 30 proteins showed a lower abundance in E2-treated NT2/D1 cancer cells. Functional analysis using IPA highlighted some activation processes such as migration, invasion, metastasis, and tumor growth. Interestingly, the treatment with E2 and ERβ-selective agonist DPN increased the migration of NT2/D1 cells. On the other hand, ERα-selective agonist PPT did not modify cell migration, indicating that ERβ is the upstream …

Tiphaine Monsinjon

Tiphaine Monsinjon

Université du Havre

The Journal of Steroid Biochemistry and Molecular Biology

Evolutionary history and functional characterization of duplicated G protein-coupled estrogen receptors in European sea bass

Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish …

Alessandro Prete

Alessandro Prete

University of Birmingham

The Journal of Steroid Biochemistry and Molecular Biology

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5–10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and …