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。人类基因组计划揭示了人类有48种不同的核受体（nr）。内分泌干扰物可与所有这些nr结合，因此应检查这些化学品对nr的影响。制备“构象变化敏感抗体”，区分nr的活性和非活性构象，我们开发了一种测定方法来估计受体结合能力和化学物质的激活能力。该方法基于配体结合引起的NR从无活性载脂蛋白构象转变为活性全息构象的构象变化量。在本研究中，我们试图将此方法应用于所有48例NRs。本文首先以III类类固醇激素受体为研究对象，采用“定量进化轨迹分析”的方法对48个人类rna进行了分析，重点探讨了它们的结构-功能关系。分析清楚地表明，有两种不同类型的核磁共振。一种是通常的配体诱导激活机制，而另一种是自发的“自激活”机制。后一种机制的雌激素相关受体γ (ERR_γ)已表明它特异性结合双酚A (BPA)，一种被认为是内分泌干扰物的成员。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测定方法，该方法甚至适用于自激活型核糖核酸。目前的研究已经取得了显著的成果，例如发现了自激活型的rna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.