RATIONALE The most frequently occurring phthalate, di(2-ethylhexyl) phthalate (DEHP), causes adverse effects on glucose homeostasis and insulin sensitivity in several cell models and epidemiological studies. However, thus far, there is no information available on the molecular interaction of phthalates and one of the key regulators of the metabolism, the peroxisome proliferator-activated receptor gamma (PPARγ). Since the endogenous ligand of PPARγ, 15-deoxy-delta-12,14-prostaglandin J2 (15Δ-PGJ2 ), features structural similarity to DEHP and its main metabolites produced in human hepatic metabolism, mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), we tested the hypothesis of direct interactions between PPARγ and DEHP or its transformation products. METHODS Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and docking were conducted to obtain structural insights into the interactions and surface plasmon resonance (SPR) analysis to reveal information about binding levels. To confirm the activation of PPARγ upon ligand binding on the cellular level, the GeneBLAzer® bioassay was performed. RESULTS HDX-MS and SPR analyses demonstrated that the metabolites MEHP and MEOHP, but not DEHP itself, bind to the ligand binding pocket of PPARγ. This binding leads to typical activation-associated conformational changes, as observed with its endogenous ligand 15Δ-PGJ2 . Furthermore, the reporter gene assay confirmed productive interaction. DEHP was inactive up to a concentration of 14 μM, while the metabolites MEHP and MEOHP were active at low micromolar concentrations. CONCLUSIONS In summary, this study gives structural insights into the direct interaction of PPARγ with MEHP and MEOHP and shows that the DEHP transformation products may modulate the lipid metabolism through PPARγ pathways.

中文翻译：

---

邻苯二甲酸单（2-乙基己基）邻苯二甲酸酯（MEHP）和邻苯二甲酸单（2-乙基-5-氧己基）邻苯二甲酸酯（MEOHP），但邻苯二甲酸二（2-乙基己基）邻苯二甲酸酯（DEHP）不能与过氧化物酶体增殖物激活的受体γ有效结合。

理由在几种细胞模型和流行病学研究中，最常出现的邻苯二甲酸酯邻苯二甲酸二（2-乙基己基）酯（DEHP）对葡萄糖稳态和胰岛素敏感性产生不利影响。然而，到目前为止，还没有关于邻苯二甲酸酯与新陈代谢的关键调节剂之一-过氧化物酶体增殖物激活受体γ（PPARγ）的分子相互作用的信息。由于PPARγ的内源性配体15-脱氧-δ12,14-前列腺素J2（15Δ-PGJ2）具有与DEHP及其在人体肝脏代谢中产生的主要代谢产物的结构相似性，因此邻苯二甲酸单（2-乙基己基）酯（MEHP）和邻苯二甲酸单（2-乙基-5-氧己基）酯（MEOHP），我们测试了PPARγ与DEHP或其转化产物之间直接相互作用的假设。方法进行氢/氘交换质谱（HDX-MS）和对接，以了解相互作用和表面等离振子共振（SPR）分析的结构见解，以揭示有关结合水平的信息。为了确认配体在细胞水平上结合后PPARγ的活化，进行了生物测定。结果HDX-MS和SPR分析表明，代谢产物MEHP和MEOHP结合了PPARγ的配体结合口袋，但不结合DEHP本身。如其内源配体15Δ-PGJ2所观察到的，这种结合导致典型的激活相关构象变化。此外，报告基因测定证实了生产性相互作用。浓度高达14μM时，DEHP无活性，而代谢物MEHP和MEOHP在低微摩尔浓度下具有活性。结论总而言之，