Risk assessment of endocrine disruptors in nuclear receptors by means of antibody-sensing method to measure the conformation change associated with ligand binding

被认为是内分泌干扰物的化学品主要通过与雌激素受体（ER）和/或雄激素受体（AR）结合来扰乱内分泌激素系统。人类基因组计划发现，人类有48种不同的核受体。内分泌干扰物可与所有这些NR结合，因此应检查这些NR是否受此类化学品的影响。制备区分NR的活性和非活性构象的“构象变化传感抗体”，我们已经开发了一种测定方法来估计化学品的受体结合能力和活化能力。该方法基于配体结合诱导的构象变化的量，所述构象变化导致NR从非活性脱辅基构象变化为活性全构象。在本研究中，我们尝试将这一方法应用于所有48个核受体，主要针对III组类固醇激素受体，首先利用建立的“定量进化痕迹分析”方法对48个人类核受体进行了分析，特别是探讨了结构与功能的关系。分析清楚地表明，有两种不同类型的NR。一种是在通常的配体诱导的激活机制，而另一种是在自发的“自激活”机制。雌激素相关受体γ（ERR_γ）与内分泌干扰物双酚A（BPA）特异性结合。虽然BPA被认为与ER和AR结合，但ERR_γ被发现是BPA的真正受体。我们成功地开发了一种新的构象变化传感检测方法，该方法甚至适用于自激活类型的NR。本研究取得了一些重要成果，如发现了自激活型NR和ERR_γ作为内分泌干扰物BPA的受体。

The chemicals acknowledged as endocrine-disruptors are believed to perturb an endocrine hormone system mainly by binding to the estrogen receptor (ER) and/or androgen receptor (AR). The human genome project revealed that there are 48 different kinds of nuclear receptors (NRs) in human. Endocrine disruptors are feasible to bind to all of these NRs, and thus these NRs should be examined for the influence of such chemicals. Preparing 'conformation change-sensing antibodies' that distinguish the active and inactive conformations of NRs, we have developed an assay methodology to estimate the receptor binding ability and activation capability of chemicals. This mehod is based on the amount of conformation change induced by ligand binding that causes a change of the NR from inactive apo conformation to active holo conformation. In the present study, we attempted to develop this methodology to all of 48 NRs.Mainly focusing on group III steroid hormone receptors, we first analyzed 48 human NRs by means of the method established as the 'quantitative evolutionary trace analysis' especially to explore the structure-function relationships. The analyses clearly revealed that there are two distinct types of NRs. One type is in a usual ligand-induced activation mechanism, while the other is in a spontaneous 'self-activation' mechanism. Estrogen-related receptor γ (ERR_γ) of the latter mechanism has shown that it binds specifically bisphenol A (BPA), a putative member of endocrine disruptors. Although BPA has been believed to bind to ER and AR, ERR_γ was found to be a BPA's true receptor. We succeeded in development of a novel conformation-change sensing assay method, which is applicable to even a self-activation type of NRs. The present study has led to significant fruitful results such as the discovery of self-activation type of NRs and ERR_γ as the receptor of an endocrine disruptor BPA.