Choi Gyuho, Kang Hyunkoo, Suh Jung-Soo, Lee Haksoo, Han Kiseok, Yoo Gaeun, Jo Hyejin, Shin Yeong Min, Kim Tae-Jin, Youn BuHyun
Department of Integrated Biological Science, Pusan National University, Busan 46241, Republic of Korea.
Food Safety Risk Assessment Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea.
Biomater Res. 2024 Mar 7;28:0010. doi: 10.34133/bmr.0010. eCollection 2024.
The increasing prevalence of endocrine-disrupting chemicals (EDCs) in our environment is a growing concern, with numerous studies highlighting their adverse effects on the human endocrine system. Among the EDCs, estrogenic endocrine-disrupting chemicals (eEDCs) are exogenous compounds that perturb estrogenic hormone function by interfering with estrogen receptor (ER) homo (α/α, β/β) or hetero (α/β) dimerization. To date, a comprehensive screening approach for eEDCs affecting all ER dimer forms in live cells is lacking. Here, we developed ER dimerization-detecting biosensors (ERDDBs), based on bioluminescence resonance energy transfer, for dimerization detection and rapid eEDC identification. To enhance the performance of these biosensors, we determined optimal donor and acceptor locations using computational analysis. Additionally, employing HaloTag as the acceptor and incorporating the P2A peptide as a linker yielded the highest sensitivity among the prototypes. We also established stable cell lines to screen potential ER dimerization inducers among estrogen analogs (EAs). The EAs were categorized through cross-comparison of ER dimer responses, utilizing EC values derived from a standard curve established with 17β-estradiol. We successfully classified 26 of 72 EAs, identifying which ER dimerization types they induce. Overall, our study underscores the effectiveness of the optimized ERDDB for detecting ER dimerization and its applicability in screening and identifying eEDCs.
环境中内分泌干扰化学物质（EDCs）的日益普遍令人愈发担忧，众多研究都强调了它们对人体内分泌系统的不利影响。在EDCs中，雌激素类内分泌干扰化学物质（eEDCs）是外源性化合物，通过干扰雌激素受体（ER）的同型（α/α、β/β）或异型（α/β）二聚化来扰乱雌激素功能。迄今为止，尚缺乏一种能在活细胞中全面筛选影响所有ER二聚体形式的eEDCs的方法。在此，我们基于生物发光共振能量转移开发了用于二聚化检测和快速鉴定eEDCs的ER二聚化检测生物传感器（ERDDBs）。为提高这些生物传感器的性能，我们通过计算分析确定了最佳供体和受体位置。此外，使用HaloTag作为受体并引入P2A肽作为连接子，在原型中产生了最高的灵敏度。我们还建立了稳定细胞系，以筛选雌激素类似物（EAs）中潜在的ER二聚化诱导剂。通过对ER二聚体反应进行交叉比较，利用由17β - 雌二醇建立的标准曲线得出的EC值对EAs进行分类。我们成功对72种EAs中的26种进行了分类，确定了它们诱导的ER二聚化类型。总体而言，我们的研究强调了优化后的ERDDB在检测ER二聚化方面的有效性及其在筛选和鉴定eEDCs中的适用性。
