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Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor-gamma.

Okada H, Tokunaga T, Liu X, Takayanagi S, Matsushima A, Shimohigashi Y - Environ. Health Perspect. (2008)

Bottom Line: When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding.The maximal activity was attained when one of the methyl groups was removed.These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, The Research-Education Centre of Risk Science, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT

Background: Various lines of evidence have shown that bisphenol A [BPA; HO-C6H4-C(CH3)2-C6H4-OH] acts as an endocrine disruptor when present in very low doses. We have recently demonstrated that BPA binds strongly to human estrogen-related receptor-gamma (ERR-gamma ) in a binding assay using [3H]4-hydroxytamoxifen ([3H]4-OHT). We also demonstrated that BPA inhibits the deactivation activity of 4-OHT.

Objectives: In the present study, we intended to obtain direct evidence that BPA interacts with ERR-gamma as a strong binder, and also to clarify the structural requirements of BPA for its binding to ERR-gamma.

Methods: We examined [3H]BPA in the saturation binding assay using the ligand binding domain of ERR-gamma and analyzed the result using Scatchard plot analysis. A number of BPA derivatives were tested in the competitive binding assay using [3H]BPA as a tracer and in the luciferase reporter gene assay.

Results: [3H]BPA showed a KD of 5.50 nM at a Bmax of 14.4 nmol/mg. When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding. The maximal activity was attained when one of the methyl groups was removed. All of the potent BPA derivatives retained a high constitutive basal activity of ERR-gamma in the luciferase reporter gene assay and exhibited a distinct inhibitory activity against 4-OHT.

Conclusion: These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

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Related in: MedlinePlus

The saturation binding analysis of BPA for ERR-γ . (A) Saturation binding curve of [3H]BPA for the recombinant human ERR-γ –LBD showing total, nonspecific, and specific binding. Determination of nonspecific binding was carried out by excess unlabeled BPA (10 μM). (B) Binding data analyzed by Scatchard plot analysis to estimate the dissociation constant (KD) and the receptor density (Bmax). The plot was linear, the KD value was estimated to be 5.50 ± 0.87 nM, and Bmax was 14.4 nmol/mg protein. The saturation binding analysis was performed in duplicate and repeated four times.
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f1-ehp0116-000032: The saturation binding analysis of BPA for ERR-γ . (A) Saturation binding curve of [3H]BPA for the recombinant human ERR-γ –LBD showing total, nonspecific, and specific binding. Determination of nonspecific binding was carried out by excess unlabeled BPA (10 μM). (B) Binding data analyzed by Scatchard plot analysis to estimate the dissociation constant (KD) and the receptor density (Bmax). The plot was linear, the KD value was estimated to be 5.50 ± 0.87 nM, and Bmax was 14.4 nmol/mg protein. The saturation binding analysis was performed in duplicate and repeated four times.

Mentions: As shown in Figure 1A, the binding of BPA to ERR-γ was specific and saturated. Specific binding of [3H]BPA to ERR-γ was estimated to be approximately 80%, which we judged to be a very high value. In other words, the level of nonspecific binding of [3H]BPA was very low (Figure 1A). The high level of specific binding of [3H]BPA clearly demonstrated that BPA has no structural elements for nonspecific binding to the receptor protein and exclusively occupies the binding pocket of ERR-γ –LBD. GST did not bind [3H]BPA at all. It should be noted that the specific binding of [3H]4-OHT was only about 50% (Takayanagi et al. 2006).


Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor-gamma.

Okada H, Tokunaga T, Liu X, Takayanagi S, Matsushima A, Shimohigashi Y - Environ. Health Perspect. (2008)

The saturation binding analysis of BPA for ERR-γ . (A) Saturation binding curve of [3H]BPA for the recombinant human ERR-γ –LBD showing total, nonspecific, and specific binding. Determination of nonspecific binding was carried out by excess unlabeled BPA (10 μM). (B) Binding data analyzed by Scatchard plot analysis to estimate the dissociation constant (KD) and the receptor density (Bmax). The plot was linear, the KD value was estimated to be 5.50 ± 0.87 nM, and Bmax was 14.4 nmol/mg protein. The saturation binding analysis was performed in duplicate and repeated four times.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2199305&req=5

f1-ehp0116-000032: The saturation binding analysis of BPA for ERR-γ . (A) Saturation binding curve of [3H]BPA for the recombinant human ERR-γ –LBD showing total, nonspecific, and specific binding. Determination of nonspecific binding was carried out by excess unlabeled BPA (10 μM). (B) Binding data analyzed by Scatchard plot analysis to estimate the dissociation constant (KD) and the receptor density (Bmax). The plot was linear, the KD value was estimated to be 5.50 ± 0.87 nM, and Bmax was 14.4 nmol/mg protein. The saturation binding analysis was performed in duplicate and repeated four times.
Mentions: As shown in Figure 1A, the binding of BPA to ERR-γ was specific and saturated. Specific binding of [3H]BPA to ERR-γ was estimated to be approximately 80%, which we judged to be a very high value. In other words, the level of nonspecific binding of [3H]BPA was very low (Figure 1A). The high level of specific binding of [3H]BPA clearly demonstrated that BPA has no structural elements for nonspecific binding to the receptor protein and exclusively occupies the binding pocket of ERR-γ –LBD. GST did not bind [3H]BPA at all. It should be noted that the specific binding of [3H]4-OHT was only about 50% (Takayanagi et al. 2006).

Bottom Line: When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding.The maximal activity was attained when one of the methyl groups was removed.These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, The Research-Education Centre of Risk Science, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT

Background: Various lines of evidence have shown that bisphenol A [BPA; HO-C6H4-C(CH3)2-C6H4-OH] acts as an endocrine disruptor when present in very low doses. We have recently demonstrated that BPA binds strongly to human estrogen-related receptor-gamma (ERR-gamma ) in a binding assay using [3H]4-hydroxytamoxifen ([3H]4-OHT). We also demonstrated that BPA inhibits the deactivation activity of 4-OHT.

Objectives: In the present study, we intended to obtain direct evidence that BPA interacts with ERR-gamma as a strong binder, and also to clarify the structural requirements of BPA for its binding to ERR-gamma.

Methods: We examined [3H]BPA in the saturation binding assay using the ligand binding domain of ERR-gamma and analyzed the result using Scatchard plot analysis. A number of BPA derivatives were tested in the competitive binding assay using [3H]BPA as a tracer and in the luciferase reporter gene assay.

Results: [3H]BPA showed a KD of 5.50 nM at a Bmax of 14.4 nmol/mg. When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-gamma , we found that only one of the two phenol-hydroxyl groups was essential for the full binding. The maximal activity was attained when one of the methyl groups was removed. All of the potent BPA derivatives retained a high constitutive basal activity of ERR-gamma in the luciferase reporter gene assay and exhibited a distinct inhibitory activity against 4-OHT.

Conclusion: These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-gamma.

Show MeSH
Related in: MedlinePlus