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

这些被认为是内分泌干扰物的化学物质被认为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。人类基因组计划显示，人类体内有48种不同的核受体。内分泌干扰物与所有这些NRs结合是可行的，因此应该检查这些NRs是否受到这些化学物质的影响。为了制备区分NRs活性构象和非活性构象的“构象变化感知抗体”，我们发展了一种测定方法来估计化学物质的受体结合能力和激活能力。这种方法是基于配体结合引起的构象变化量，从而导致NR从非活性脱辅基构象到活性全息构象的变化。在本研究中，我们试图将这一方法发展到所有的48个NRs上。我们首先以III族类固醇激素受体为主要研究对象，通过建立定量进化轨迹分析的方法对48个NRs进行分析，特别是探索其结构与功能的关系。分析清楚地表明，有两种截然不同的NRS类型。一种是常见的配体诱导的激活机制，另一种是自发的自激活机制。雌激素相关受体γ(ERR_γ)是后一种机制中的一种，它与内分泌干扰物中的双酚A(BPA)特异性结合。虽然双酚A被认为与ER和AR结合，但ERR_γ被发现是双酚A的真正受体。我们成功地开发了一种新的构象变化传感分析方法，该方法甚至适用于自激活类型的NRS。本研究取得了丰硕的成果，如发现了自激活型NRs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.