SCREENS TO IDENTIFY GAIN OF FUNCTION AH RECEPTOR MUTANTS INVOLVED IN DEGRADATION

描述(申请人提供)：AH受体(AHR)是一种配体激活的转录因子，属于碱性-螺旋-环-螺旋/每-Arnt-SIM(bHLH/PAS)蛋白家族。在C57BL/6J小鼠模型系统中，已对AHR介导的信号转导进行了广泛的研究，试图定义该途径的组成部分，了解蛋白质和DNA的相互作用，并确定可能影响人类健康的基因表达的特定变化。人们对AHR蛋白的降解重视较少，尤其是与关闭AHR信号通路有关的。不幸的是，调节AHR降解的蛋白质和AHR降解所必需的结构域仍有待鉴定。因此，必须采用多方面的方法来确定参与降解过程的候选靶酶，并获得AHR的“功能获得”突变体。因此，这一建议的中心假设是，蛋白质降解的缺陷将表现为AHR的“功能获得”突变。提出了三组研究来解决这一假说，并分离出AHR途径中第一个配体激活的功能突变体的增益。首先，将开展利用细菌双杂交筛选来鉴定与AHR的COOH-末端结构域相互作用的蛋白质的研究。其次，表达稳定整合的绿色荧光蛋白标记的AHR的细胞系将用于对不降解AHR的细胞进行遗传筛选。最后，将产生表达功能性AHR信号通路的酵母菌株。参与蛋白质降解的特定基因将从这些菌株中删除，以评估它们在AHR降解中的作用。因此，拟议的实验利用候选基因方法和遗传筛选，并使用哺乳动物细胞、酵母和细菌来解决这一假设。对于不容易从生物体中代谢或清除的卤代芳香化合物来说，关闭AHR介导的信号转导和这一途径的调节机制尤其关键。此外，由于AHR信号的建模和风险评估在很大程度上依赖于了解完整的信号通路，因此确定AHR通路是如何衰减的是至关重要的。相关声明：暴露在环境污染物中会对人类健康造成不利影响。对于通过与内源蛋白结合和基因调控发挥作用的化学物质，了解这些途径是如何调控的是至关重要的。了解ah受体蛋白是如何降解的，对于以2，3，7，8，四氯二苯偶氮-对二恶英(Dioxin)为代表的化学物质的风险评估至关重要，并将适用于其他毒理学相关的受体(即类固醇激素受体)。

DESCRIPTION (provided by applicant): The Ah-receptor (AHR) is a ligand activated transcription factor that belongs to the family of basic-helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) proteins. AHR-mediated signaling has been extensively investigated in the C57BL/6J mouse model system in an attempt to define the components of the pathway, understand protein and DNA interactions and define specific changes in gene expression that likely impact human health. There has been less emphasis placed on the degradation of AHR protein especially as it relates turning the AHR signaling pathway off. Unfortunately, the proteins that modulate the degradation of the AHR and the domains of the AHR that are necessary for degradation remain to be identified. Thus, multifaceted approaches must be initiated to define candidate target enzymes involved in the degradation process and obtain "gain of function" mutants of the AHR. Therefore, the central hypothesis of this proposal is that defects in protein degradation will be manifest as "gain of function" mutants of the AHR. Three sets of studies are proposed to address this hypothesis and isolate the first ligand- activated gain of function mutants in the AHR pathway. First, studies will be carried out that utilize a bacterial two-hybrid screen to identify proteins interacting with the COOH-terminal domain of the AHR. Second, cell lines expressing a stably integrated GFP tagged AHR will be used to carry out a genetic screen for cells that do not degrade the AHR. Finally, yeast strains will be generated that express a functional AHR signaling pathway. Specific genes involved in protein degradation will be deleted from these strains to assess their role in AHR degradation. Thus, the proposed experiments utilize candidate gene approaches as well as genetic screens and employ mammalian cells, yeast and bacteria to address this hypothesis. The mechanism involved in turning off AHR-mediated signaling and the regulation of this pathway are especially critical with respect to halogenated aromatic compounds that are not readily metabolized or cleared from an organism. In addition, since modeling of AHR signaling and risk assessment in humans relies heavily on understanding the complete signaling pathway, it is essential to determine how the AHR pathway is attenuated. Statement of Relevance: Human health can be adversely affected by exposure to environmental contaminants. For chemicals that act through association with endogenous proteins and modulation of gene regulation, it is essential to understand how the pathways are regulated. Understanding how the Ah receptor protein is degraded is critical to risk assessment of chemicals typified by 2,3,7,8, tetrachlorodibezo-p-dioxin (Dioxin), and will be applicable to other toxicologically relevant receptors (i.e. steroid hormone receptors).