Studies on the Ah Receptors Signaling Mechanism

说明（由申请方提供）：商业和天然产生的二恶英是氯化多环芳烃，是剧毒环境污染物。已知这些物质是啮齿类动物的强效致癌物和疑似人类致癌物。这类药剂最著名的原型是2，3，7，8-四氯二苯并对二恶英（TCDD）。它已被充分证明，大多数，如果不是全部，TCDD的影响是通过介导的Ah受体（AhR）。为了更好地理解TCDD的作用机制，我们将研究AhR信号通路的分子机制。工作假设如下：TCDD结合后，受体发生核转位，AhR在核内形成AhR/Arnt/DRE复合物，导致基因转录激活。我们发现p23和CyP 40在体外增强了这种AhR三元复合物的形成（参考文献：1.谢蒂，P. V.，王，X.，和Chan，W. K.（2004）Arch. Biochem. Biophys. 42 - 9，42-9; 2.谢蒂，P. V.，巴格瓦特，B。是的，和Chan，W. K.等人（2003）Biochem. Pharmacol. 65，941-8），并且这些蛋白质似乎在细胞培养研究中影响AhR信号传导。本研究的重点是p23和CyP 40在AhR信号转导中的内源性作用。本研究的主要目的如下：我们将利用p23和CyP 40基因敲低和过表达的细胞来确定（1）在完整细胞中，p23和CyP 40是否影响AhR和Arnt的异源二聚体化以及异源二聚体与DRE的结合（目的1）;在完整细胞中，在基因转录激活之前，AhR复合物与增强子区域的组装受到p23和CyP 40的影响（Aim 2）和（3）细胞核AhR的命运受p23和CyP 40的影响。我们还将研究p23和CyP 40在AhR信号传导中的需求（目的4）。将进行缺失和突变研究，以确定AhR功能对p23和CyP 40的最低结构要求。将检查和表征CyP 40和AhR之间的相互作用。CyP 40相互作用的蛋白质是必不可少的CyP 40对AhR功能的影响将被确定和表征。

DESCRIPTION (provided by applicant): Dioxins, generated both commercially and naturally, are chlorinated polycyclic aromatic hydrocarbons that are highly toxic environmental contaminants. These agents are known to be potent rodent carcinogens and suspected human carcinogens. The best known prototype of this group of agents is 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). It has been well-documented that most, if not all, of the TCDD effects are mediated through the Ah receptor (AhR). In an effort to better understand the mechanism of TCDD action, we will investigate the molecular mechanism of the AhR signaling pathway. The working hypothesis is as follows: Upon TCDD binding, nuclear translocation of the receptor occurs and the AhR forms the AhR/Arnt/DRE complex in the nucleus, leading to activation of gene transcription. We discovered that p23 and CyP40 potentiate the formation of this AhR ternary complex in vitro (Refs: 1. Shetty, P. V., Wang, X., and Chan, W. K. (2004) Arch. Biochem. Biophys. 429, 42-9; 2. Shetty, P. V., Bhagwat, B. Y., and Chan, W. K. (2003) Biochem. Pharmacol. 65, 941-8) and these proteins appear to affect the AhR signaling in cell culture studies. This proposal focuses on the endogenous roles of p23 and CyP40 in the AhR signaling. Four specific aims have been proposed as follows: We will use p23 and CyP40 knockdown and overexpressed cells to determine whether (1) the heterodimerization of AhR and Arnt and the binding of the heterodimer to the DRE are affected by p23 and CyP40 in intact cells (Aim 1); the assembly of the AhR complex to the enhancer region prior to activation of gene transcription is affected by p23 and CyP40 in intact cells (Aim 2) and (3) the fate of the nuclear AhR is affected by p23 and CyP40. We will also examine the requirements of p23 and CyP40 in the AhR signaling (Aim 4). Deletion and mutation studies will be performed to map out the minimal structural requirement of p23 and CyP40 for the AhR function. Interactions between CyP40 and AhR will be examined and characterized. CyP40-interacting proteins that are essential for the full CyP40 effect on the AhR function will be identified and characterized.