Elsevier

Toxicology in Vitro

Volume 44, October 2017, Pages 287-302
Toxicology in Vitro

Binding of bisphenol A, bisphenol AF, and bisphenol S on the androgen receptor: Coregulator recruitment and stimulation of potential interaction sites

https://doi.org/10.1016/j.tiv.2017.07.020Get rights and content

Highlights

  • Coregulator peptides recruited by BPA-AR are different from agonist R1881-AR.

  • BPA- and BPAF-AR recruited similar coregulators while BPS-AR recruited less.

  • Molecular dynamics study showed multiple binding sites for BPA analogues-AR.

  • BPA analogues can modify the AR binding surface for coregulator interactions.

Abstract

Bisphenol A (BPA), bisphenol AF (BPAF), and bisphenol S (BPS) are well known endocrine disruptors. Previous in vitro studies showed that these compounds antagonize androgen receptor (AR) transcriptional activity; however, the mechanisms of action are unclear. In the present study, we investigated interactions of coregulator peptides with BPA, BPAF, or BPS at the AR complexes using Micro Array for Real-time Coregulator Nuclear Receptor Interaction (MARCoNI) assays and assessed the binding of these compounds on AR by molecular dynamics (MD) simulations. The set of coregulator peptides that are recruited by BPA-bound AR, either positively/or negatively, are different from those recruited by the agonist R1881-bound AR. Therefore, the data indicates that BPA shows no similarities to R1881 and suggests that it may recruit other coregulators to the AR complex. BPAF-bound AR recruits about 70–80% of the same coregulator peptides as BPA-bound AR. Meanwhile, BPS-bound AR interacts with only few peptides compared to BPA or BPAF-bound AR. MD results show that multiple binding sites with varying binding affinities are available on AR for BPA, BPAF, and BPS, indicating the availability of modified binding surfaces on AR for coregulator interactions. These findings help explain some of the distinct AR-related toxicities observed with bisphenol chemicals and raise concern for the use of substitutes for BPA in commercial products.

Introduction

Bisphenols (BPA, BPAF and BPS) are reported to be endocrine disruptor chemicals (EDCs), causing a variety of human health issues (Rochester, 2013, Rochester and Bolden, 2015). The vast majority of studies have focused on the agonistic effects of BPA analogues on the estrogen-signaling pathway because they can bind to the estrogen receptors alpha and beta (ERα and β) and activate estrogen responsive genes. Contrary to the positive modulation on ER, BPA analogues antagonize the transcriptional activity of the androgen receptor (AR), which could affect the AR signaling pathway (Safe, 2002, Teng et al., 2013, Rehan et al., 2015).

Androgens, upon binding to the AR, play a major role in male sex organ development and are critical for maintaining male reproductive function (Luccio-Camelo and Prins, 2011, Sidorkiwicz et al., 2017). In addition, they affect normal physiology of a wide variety of tissues and deregulate AR function, which may potentially lead to cancer (Rochester, 2013, Tan et al., 2015, Dai et al., 2017).

The AR is a member of the nuclear receptor superfamily and is consistent with the characteristic nuclear receptor modular structure (Helsen and Claessens, 2014). However, the 538 amino acid stretch at the N-terminus domain bears (NTD) little homology with other nuclear receptors, showing an intrinsically disordered structure, which is in contrast to the DNA-binding domain (DBD) and ligand-binding domain (LBD) (McEwan, 2012). Ligand binding to the C-terminal of the AR leads to conformation changes and creates two protein-protein interaction surfaces for coregulators to bind to the AF2 and BF3 sites. Importantly, the BF3 site can bind small molecules and allosterically regulate protein-protein interactions at the AF2 site (Estebanez-Perpina et al., 2007). Furthermore, mutations at this location occur frequently in prostate cancer patients (Munuganti et al., 2014). The functional activity of the ligand-bound AR requires interactions between the N- and C- terminus of the receptor protein as well as protein-protein interactions with coregulators (Kumar and McEwan, 2012). Based on cell types, target genes, and the types of ligands bound to the receptor, transcription of the target genes could become enhanced or repressed, depending on the coregulator or corepressor. (Millard et al., 2013, O'Malley and McKenna, 2008). Hundreds of nuclear receptor (NR) coregulators have been identified (http://www.NURSA.org) and many have been revealed with the proteomic analysis approach (Foulds et al., 2013). The transcription levels of NR target genes are dictated by the coregulator proteins that are recruited to the ligand bound NR. This, in turn, modifies the chromatin structure to either favor transcriptional activation or repression (Millard et al., 2013). The conserved interaction motifs of the coregulators and NR, with or without ligands, are LxxLL (the NR box; (Heery et al., 1997)) and LxxH/IIxxxI/L (CoRNR box; (Hu and Lazar, 1999, Nagy et al., 1999)). In general, coregulators possess enzymatic activity, which modifies chromatin structure via various mechanisms and influences the rate of transcription (Millard et al., 2013).

In the present study, the objective was to search for corregulator protein recruitment by the AR when BPA analogues were bound. We used the MARCoNI peptide screening method to identify coregulators in BPA analogue-bound AR complexes. Furthermore, we used molecular dynamic (MD) simulation techniques that have emerged as a common tool to characterize the strength of small molecule protein interactions to investigate how the interaction surfaces of the coregulators can be modified due to various ligand binding. This is the first report to describe the recruitment of coregulators to the BPA analogue-AR complexes and to show that the BPA analogues may have the potential to bind at multiple locations on the AR complex.

Section snippets

Reagents

The synthetic androgen, methyltrienolone (R1881, CASRN: 965-93-5), was purchased from RTI International (Durham, NC). The three EDCs, BPA (bisphenol A, Chemical Abstracts Services Registry Number, CASRN: 80-05-7), BPAF (bisphenol AF, CASRN: 1478-61-1), BPS (bisphenol S, CASRN: 80-08-1), and the synthetic anti-androgen CPA (cyproterone acetate, CASRN: 2098-66-0) were obtained from the Midwest Research Institute (Kansas City, MO) through a contract with the National Toxicology Program. DHT

Results

Previous studies showed that BPA represses androgen-induced reporter activity in transiently transfected CV-1 cells through multiple molecular mechanisms (Teng et al., 2013). To understand the coregulator interactions with BPA analogues bound to the AR, a MARCoNI assay was applied. This assay was used to mimic NR-coregulator interactions in vitro (Wang et al., 2013). The MARCoNI assay consists of 154 immobilized coregulator-derived binding peptides, including 129 highly conserved helical LxxLL

Discussion

Dynamic communication between the structure of the ligand-bound AR and the regulator recruitment has been found to be crucial for its function (He and Wilson, 2003, Xu et al., 2011). In the present study, the coregulator recruitment of BPA analogues was compared with that of R1881 (agonist) and CPA (antagonist) when bound to the AR. We found that there were significant differences in recruited coregulators between BPA analogues and R1881 or CPA. In addition, results from MD simulations of AR

Conflict of interests

The authors declare that they have no conflict of interests.

Transparency document

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Acknowledgments

We thank Vicki Sutherland, Lars Pedersen, and Jeffrey Tyler Ramsey for critical review. Research support was provided by Intramural Research Programs of the National Toxicology Program, National Institute of Environmental Health sciences (NIEHS), National Institutes of Health (NIH), and contract with PamGene International. This research was also supported by Research Project Number Z01-ES043010 (L.P) in the Intramural Research Program of NIEHS, NIH.

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