scholarly journals Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H

2019 ◽  
Author(s):  
Shaheena Parween ◽  
Giovanna DiNardo ◽  
Francesca Baj ◽  
Chao Zhang ◽  
Gianfranco Gilardi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Treatment Options ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Therapeutic Interventions ◽  
Diagnostic Tools ◽  
Variant Form ◽  
Aromatase Activity ◽  
Allelic Variants ◽  
P450 Oxidoreductase ◽  
Redox Partner

AbstractAromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, result in a reduction of CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284L as well as the POR-P284T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained around 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing towards a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point towards a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284L, P284T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in both the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis.

scholarly journals Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction

Antioxidants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 673
Author(s):  
Masakazu Sugishima ◽  
Junichi Taira ◽  
Tatsuya Sagara ◽  
Ryota Nakao ◽  
Hideaki Sato ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Enzymatic Activity ◽  
Heme Oxygenase ◽  
Analytical Ultracentrifugation ◽  
Dynamic Equilibrium ◽  
P450 Oxidoreductase ◽  
Nadph Cytochrome ◽  
Redox Partner ◽  
Cytochrome P450 Oxidoreductase

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme–HO-1, whereas the reduction in heme–HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme–HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme–HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme–HO-1 in solution. We conclude that the “open–close” transition of CPR is indispensable for electron transfer from CPR to heme–HO-1.

scholarly journals Loss of multiple enzyme activities due to the human genetic variation P284T in NADPH cytochrome P450 oxidoreductase

2019 ◽  
Author(s):  
Shaheena Parween ◽  
Maria Natalia Rojas Velazquez ◽  
Sameer S. Udhane ◽  
Norio Kagawa ◽  
Amit V. Pandey
Keyword(s):  
Cytochrome P450 ◽  
Cytochrome C ◽  
Negative Impact ◽  
Aromatase Activity ◽  
Cytochrome P450 Enzymes ◽  
Ferricyanide Reduction ◽  
P450 Oxidoreductase ◽  
Nadph Cytochrome ◽  
Reduction Activity ◽  
Cytochrome P450 Oxidoreductase

AbstractCytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Here we are reporting the effects of a POR genetic variant P284T which is located in the hinge region of POR that is necessary for the flexibility of domain movements. Human wild-type and P284T mutant of POR, as well as cytochrome P450 proteins, were expressed in bacteria, purified and then reconstituted in liposomes for enzyme kinetic assays. Quality of POR proteins was checked by cytochrome c, ferricyanide and tetrazolium dye reduction assay and measurements flavin content. We found that for the P284T variant of POR the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4 and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation.

scholarly journals Regulation of Microsomal P450, Redox Partner Proteins, and Steroidogenesis in the Developing Testes of the Neonatal Pig

Endocrinology ◽  
2002 ◽  
Vol 143 (9) ◽  
pp. 3361-3369 ◽  
Author(s):  
F. M. Moran ◽  
J. J. Ford ◽  
C. J. Corbin ◽  
S. M. Mapes ◽  
V. C. Njar ◽  
...  
Keyword(s):  
Gnrh Antagonist ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reduced Form ◽  
Testicular Development ◽  
Aromatase Activity ◽  
P450 Reductase ◽  
Redox Partner ◽  
Partner Proteins

Abstract Testicular growth and plasma androgen concentrations increase markedly in the first weeks of neonatal life of pigs. The regulation of steroidogenesis through this period was examined by measuring total microsomal cytochromes P450 (P450), 17α-hydroxylase/17,20-lyase P450 (P450c17) and aromatase P450 (P450arom) enzyme activities, and the redox partner proteins nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome P450 reductase (reductase) and cytochrome b5 in testicular microsomes. Testes were collected from 1–24 d of age, and testicular development was suppressed by a GnRH antagonist in some animals from d 1–14. Both 17/20-lyase and aromatase activities increased from d 1–7 but not thereafter, and 17–20-lyase activity was always at least 200-fold higher than aromatase activity. Reductase decreased in wk 1, then increased to d 24. No changes were seen in cytochrome b5 expression. GnRH antagonist treatment suppressed plasma LH, testosterone and testes growth to d 14. 17,20-Lyase and aromatase activities in testicular microsomes were reduced by 20% and 50%, respectively. Total microsomal P450 concentration was reduced by 50% on d 7, but there was no effect of treatment on reductase or cytochrome b5 expression. These data support the hypothesis that the rise in neonatal testicular androgen secretion is more likely due to gonadotropin-stimulated gonadal growth, rather than specific P450c17 expression. Neither P450c17 nor P450arom can account for the decline in total microsomal P450. Reductase and cytochrome b5 expression appears to be constitutive, but reductase levels saturate both P450c17 and P450arom.

scholarly journals Molecular Target of Endocrine Disruption in Human Luteinizing Granulosa Cells by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin: Inhibition of Estradiol Secretion Due to Decreased 17α-Hydroxylase/17,20-Lyase Cytochrome P450 Expression

Endocrinology ◽  
2003 ◽  
Vol 144 (2) ◽  
pp. 467-473 ◽  
Author(s):  
F. M. Morán ◽  
C. A. VandeVoort ◽  
J. W. Overstreet ◽  
B. L. Lasley ◽  
A. J. Conley
Keyword(s):  
Cytochrome P450 ◽  
Endocrine Disruption ◽  
Granulosa Cells ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Immunoblot Analysis ◽  
Molecular Target ◽  
Redox Partner ◽  
Lyase Activity ◽  
Tcdd Treatment ◽  
Tetrachlorodibenzo P Dioxin

Estradiol (E2) production by human luteinized granulosa cells (hLGC) is inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The molecular target of TCDD toxicity has not been identified. The decrease in E2 is ameliorated by androgen substrate addition and is not associated with changes in aromatase cytochrome P450 (P450arom) activity or protein expression. An antihuman 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) antisera and a direct radiometric assay of 17,20-lyase activity were used to test the hypothesis that TCDD targets P450c17, thereby decreasing substrate availability for E2 synthesis by hLGC. P450c17 expression and 17,20-lyase activity were detected in hLGC with high levels of E2 secretion. Western immunoblot analysis demonstrated that TCDD treatment of hLGC decreased the expression of P450c17 by as much 50% (P < 0.05). TCDD exposure induced a 65% decrease in 17,20-lyase activity (P < 0.05), but no changes were seen in P450arom or in nicotinamide adenine dinucleotide phosphate (reduced)-cytochrome P450 oxidoreductase (reductase). Furthermore, the decreases in P450c17 and 17,20-lyase were proportional to the inhibition of E2 secretion. We conclude that the molecular target for endocrine disruption of hLGC by TCDD is P450c17, specifically decreasing the supply of androgens for E2 synthesis, and that it does not involve either P450arom or the redox partner protein reductase.

scholarly journals An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Goutam Mukherjee ◽  
Prajwal P. Nandekar ◽  
Rebecca C. Wade
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Drug Targets ◽  
Catalytic Domain ◽  
Cytochrome P450 1A1 ◽  
P450 Oxidoreductase ◽  
Distal Side ◽  
Cytochrome P450 Oxidoreductase ◽  
Membrane Bound ◽  
Transient Interactions

AbstractCytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

scholarly journals B Cells and Antibodies as Targets of Therapeutic Intervention in Neuromyelitis Optica Spectrum Disorders

2021 ◽  
Vol 14 (1) ◽  
pp. 37
Author(s):  
Jan Traub ◽  
Leila Husseini ◽  
Martin S. Weber
Keyword(s):  
B Cells ◽  
Neuromyelitis Optica ◽  
Plasma Cells ◽  
Treatment Options ◽  
Treatment Strategies ◽  
Therapeutic Interventions ◽  
Complement Factor ◽  
Major Subset ◽  
Spectrum Disorders

The first description of neuromyelitis optica by Eugène Devic and Fernand Gault dates back to the 19th century, but only the discovery of aquaporin-4 autoantibodies in a major subset of affected patients in 2004 led to a fundamentally revised disease concept: Neuromyelits optica spectrum disorders (NMOSD) are now considered autoantibody-mediated autoimmune diseases, bringing the pivotal pathogenetic role of B cells and plasma cells into focus. Not long ago, there was no approved medication for this deleterious disease and off-label therapies were the only treatment options for affected patients. Within the last years, there has been a tremendous development of novel therapies with diverse treatment strategies: immunosuppression, B cell depletion, complement factor antagonism and interleukin-6 receptor blockage were shown to be effective and promising therapeutic interventions. This has led to the long-expected official approval of eculizumab in 2019 and inebilizumab in 2020. In this article, we review current pathogenetic concepts in NMOSD with a focus on the role of B cells and autoantibodies as major contributors to the propagation of these diseases. Lastly, by highlighting promising experimental and future treatment options, we aim to round up the current state of knowledge on the therapeutic arsenal in NMOSD.

scholarly journals Fructose and Mannose in Inborn Errors of Metabolism and Cancer

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 479
Author(s):  
Elizabeth L. Lieu ◽  
Neil Kelekar ◽  
Pratibha Bhalla ◽  
Jiyeon Kim
Keyword(s):  
Metabolic Disease ◽  
Inborn Errors Of Metabolism ◽  
Metabolic Diseases ◽  
Treatment Options ◽  
Biochemical Properties ◽  
Biological Molecules ◽  
Diagnostic Tools ◽  
Inborn Errors ◽  
Mannose Metabolism

History suggests that tasteful properties of sugar have been domesticated as far back as 8000 BCE. With origins in New Guinea, the cultivation of sugar quickly spread over centuries of conquest and trade. The product, which quickly integrated into common foods and onto kitchen tables, is sucrose, which is made up of glucose and fructose dimers. While sugar is commonly associated with flavor, there is a myriad of biochemical properties that explain how sugars as biological molecules function in physiological contexts. Substantial research and reviews have been done on the role of glucose in disease. This review aims to describe the role of its isomers, fructose and mannose, in the context of inborn errors of metabolism and other metabolic diseases, such as cancer. While structurally similar, fructose and mannose give rise to very differing biochemical properties and understanding these differences will guide the development of more effective therapies for metabolic disease. We will discuss pathophysiology linked to perturbations in fructose and mannose metabolism, diagnostic tools, and treatment options of the diseases.

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