Studies on the Ah Receptors Signaling Mechanism

描述（由申请人提供）：商业和自然产生的二恶英是氯化多环芳烃，是剧毒的环境污染物。已知这些物质是强效的啮齿动物致癌物和疑似的人类致癌物。这类试剂最著名的原型是2,3,7,8-四氯二苯并-对二恶英（TCDD）。有充分的证据表明，大多数（如果不是全部）TCDD效应是通过Ah受体（AhR）介导的。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，受体发生核易位，AhR在细胞核内形成AhR/Arnt/DRE复合体，导致基因转录激活。我们发现p23和CyP40在体外增强了这种AhR三元复合物的形成（参考文献：1）。王晓明、陈伟坤（2004）《文献》。物化学。生物工程学报，29,42-9；2. Shetty， P. V., Bhagwat， B. Y.和Chan， W. K.（2003）生物化学。在细胞培养研究中，这些蛋白似乎会影响AhR信号传导。本研究主要关注p23和CyP40在AhR信号传导中的内源性作用。我们将使用p23和CyP40敲低和过表达的细胞来确定(1)完整细胞中AhR和Arnt的异源二聚化以及异源二聚体与DRE的结合是否受到p23和CyP40的影响（Aim 1）；在完整细胞中，在基因转录激活之前，AhR复合物在增强子区域的组装受到p23和CyP40的影响（目的2）和(3)核AhR的命运受到p23和CyP40的影响。我们还将研究p23和CyP40在AhR信号传导中的要求（Aim 4）。将进行缺失和突变研究，以确定p23和CyP40对AhR功能的最小结构要求。CyP40和AhR之间的相互作用将被检查和表征。对CyP40对AhR功能的完全影响至关重要的CyP40相互作用蛋白将被识别和表征。

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.