Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes.

Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and ß differ from that used by 17ß-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17ß-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment.

Overexpression of mouse estrogen receptor-ß decreases but its transactivation and ligand binding domains increase the growth characteristics of E. coli.

Escherichia coli is one of the most common and widely used prokaryotic hosts for the expression of recombinant proteins. The overexpression of recombinant proteins occasionally increases bacterial growth but sometimes reduces it and becomes lethal to the host cells. Here, we report the overexpression of mouse ER-ß and its domains in the prokaryotic expression system and its opposite effect on the growth characteristics of E. coli. ER-ß protein was immunologically detected as a 53 kDa his-tag protein in the pellet of the bacterial lysate. Its overexpression, as reflected by the total protein content and expression pattern, resulted in the decrease of bacterial growth. However, the overexpression of ER-ß transactivation domain (TAD) using pIVEX and ligand binding domain (LBD) using pRSETA in E. coli BL21 (DE3) show opposite pattern. TAD was immunologically detected as 20 kDa and LBD as 22 kDa protein in the supernatant of the bacterial lysate and their overexpression increased the bacterial growth.

Nature of functional estrogen receptors at the plasma membrane.

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.

A multi-centre investigation towards reaching a consensus on the immunohistochemical detection of ERbeta in archival formalin-fixed paraffin embedded human breast tissue.

Estrogen receptor (ER) alpha is a well-established independent prognostic factor in breast cancer whose presence determines the clinical implications of adjuvant endocrine therapy. A second receptor, ERb has been described, and a number of studies have examined its expression in breast tissue. However elucidation of the role played by ERb has been hampered by published immunohistochemical studies employing a variety of protocols and scoring systems such that inter-laboratory comparisons are difficult. Here we present a multi-centre study designed to critically evaluate inter-laboratory differences in methodology. Six UK and Irish centres participated in this study. A small series of breast cancers were stained using centre-specific laboratory protocols and scored using both centrespecific and standard scoring protocols. There was generally poor agreement as to what constituted a positive or negative case when centre-specific scoring systems were used with less than half of all cases in agreement. Concordance was improved when a standard scoring system was used but varied according to threshold for positivity employed and primary antibody. Our results emphasise the need for further studies addressing the role of ERb to be based on a wider consensus on criteria for positivity. Ideally this should be based on calibration against clinical outcome.