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

Parween, Shaheena; Nardo, Giovanna Di; Baj, Francesca; Zhang, Chao; Gilardi, Gianfranco; Pandey, Amit V.

doi:10.1016/j.jsbmb.2019.105507

“…The increase obtained in the aromatase activity in the presence of imidacloprid and thiacloprid, even if not statistically significant, can be interesting to be further investigated. Indeed, these two compounds could directly act on aromatase as allosteric activators or they could exert their action indirectly on CPR that has an essential role in catalysis and acts as an effector on human aromatase conformation [80,81].…”

Section: Discussionmentioning

confidence: 99%

Molecular Basis for Endocrine Disruption by Pesticides Targeting Aromatase and Estrogen Receptor

Zhang

¹

,

Schilirò²,

Gea³

et al. 2020

IJERPH

Self Cite

View full text Add to dashboard Cite

The intensive use of pesticides has led to their increasing presence in water, soil, and agricultural products. Mounting evidence indicates that some pesticides may be endocrine disrupting chemicals (EDCs), being therefore harmful for the human health and the environment. In this study, three pesticides, glyphosate, thiacloprid, and imidacloprid, were tested for their ability to interfere with estrogen biosynthesis and/or signaling, to evaluate their potential action as EDCs. Among the tested compounds, only glyphosate inhibited aromatase activity (up to 30%) via a non-competitive inhibition or a mixed inhibition mechanism depending on the concentration applied. Then, the ability of the three pesticides to induce an estrogenic activity was tested in MELN cells. When compared to 17β-estradiol, thiacloprid and imidacloprid induced an estrogenic activity at the highest concentrations tested with a relative potency of 5.4 × 10−10 and 3.7 × 10−9, respectively. Molecular dynamics and docking simulations predicted the potential binding sites and the binding mode of the three pesticides on the structure of the two key targets, providing a rational for their mechanism as EDCs. The results demonstrate that the three pesticides are potential EDCs as glyphosate acts as an aromatase inhibitor, whereas imidacloprid and thiacloprid can interfere with estrogen induced signaling.

show abstract

“…Recombinant human aromatase was expressed in E. coli and purified as previously described [7,8]. Both wildtype and mutants were purified with the same type of buffer, i.e.…”

Section: Methodsmentioning

confidence: 99%

“…The correct activity of this protein is crucial, because alterations in the equilibrium between androgens and estrogens, hypo-or hyperproduction 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

Nardo

¹

,

Venere

²

,

Zhang

³

et al. 2021

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Recombinant human aromatase (Aro) was expressed in E. coli and purified as previously described [7,8]. Both wild-type and mutants were purified with the same type of buffer, i.e.…”

Section: Methodsmentioning

confidence: 99%

“…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.

Nardo

¹

,

Venere

²

,

Zhang

³

et al. 2021

Preprint

Self Cite

0

View full text Add to dashboard Cite

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.

show abstract

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

Cited by 23 publications

References 86 publications

Molecular Basis for Endocrine Disruption by Pesticides Targeting Aromatase and Estrogen Receptor

Molecular Basis for Endocrine Disruption by Pesticides Targeting Aromatase and Estrogen Receptor

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

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

Contact Info

Product

Resources

About