Impact of R264C and R264H polymorphisms in human aromatase function

Biotech and App Biochem

Cimicata

Baravalle

et al. 2017

Self Cite

Aromatase catalyzes the biosynthesis of estrogens from androgens. Owing to the physiological importance of this conversion of lipophilic substrates, the interaction with the lipid bilayer for this cytochrome P450 is crucial for its dynamics that must allow an easy access to substrates and inhibitors. Here, the aromatase-anastrozole interaction is studied by combining computational methods to identify possible access/egress routes with the protein inserted in the membrane and experimental tools aimed at the investigation of the effect of the inhibitor on the protein conformation. By means of molecular dynamics simulations of the protein inserted in the membrane, two channels, not detected in the starting crystal structure, are found after a 20-nSec simulation. Trypsin digestion on the recombinant protein shows that the enzyme is strongly protected by the presence of the substrate and even more by the inhibitor. DSC experiments show an increase in the melting temperature of the protein in complex with the substrate (49.3 °C) and the inhibitor (58.7 °C) compared to the ligand-free enzyme (45.9 °C), consistent with a decrease of flexibility of the protein. The inhibitor anastrozole enters the active site of the protein through a channel different from that used from the substrate and promotes a conformational change that stiffens the protein conformation and decreases the protein-protein interaction between different aromatase molecules.

Section: Methodsmentioning

confidence: 99%

Working at the membrane interface: Ligand‐induced changes in dynamic conformation and oligomeric structure in human aromatase

Biotech and App Biochem

Cimicata

Baravalle

et al. 2017

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“…The CYP19A1 deficiency is linked to genetic disorders of estrogen biosynthesis in women and developmental disorders in men [19,31,46,85,86]. Previously we have tested the CYP19A1 variants (R264C and R26H) and POR genetic variants in separate studies that focused on individual genes [59,64,70]. Cytochromes P450 and P450 reductase have a large number of genetic variations and, therefore, many individuals may harbor variations in P450 reductase as well as P450 genes that may alter the enzymatic activities of cytochromes P450 [87].…”

Section: Discussionmentioning

confidence: 99%

“…The experiments were carried out with a well-characterized recombinant soluble human CYP19A1, which has the structure and activity comparable to full-length CYP19A1 [69]. The polymorphic variants R264H and R264C of CYP19A1 were generated as described previously by Baravalle et al in 2017 [70]. The WT CYP19A1 and the two polymorphic variants, R264H and R264C, were expressed and purified based on previously described methods [69,[71][72][73].…”

Section: Recombinant Expression and Purification Of Cyp19a1 And Its Pmentioning

confidence: 99%

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

Parween

The Journal of Steroid Biochemistry and Molecular Biology

Baj

et al. 2020

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Aromatase (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 threefold 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 threefold 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.

“…The correct activity of this protein is crucial, because alterations in the equilibrium between androgens and estrogens, hypo-or hyper-production of estrogens are associated to a series of disorders that range from neurological diseases to breast cancer [5]. We have previously characterized the common R264C (rs700519) and the rarer R264H (rs2304462) polymorphic variants showing that the activity of these variants is modified by an alteration of the kinetic parameters [6,7] and by a different phosphorylation propensity, as R264 is part of a consensus sequence for phosphorylation [6]. However, this residue lies on the protein surface, being part of the G-helix and it is therefore difficult to understand how its mutation can impact the catalytic parameters only based on the location.…”

Section: Introductionmentioning

confidence: 99%

Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence.

Venere

Zhang

et al. 2021

Preprint

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Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and –bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.