Analysis of Ah Receptor Ligand Binding Specificity

Ah受体（AhR）是一种配体依赖性转录因子，已知其调节毒性和生物学活性。 各种外源化学物质的作用，这些作用是由AhR依赖性基因表达引起的。 AhR还参与内源性发育和生理过程，尽管负责的AhR基因可能是内源性的。 内源性配体未知。虽然有毒的卤代芳烃是典型的， 最高亲和力的配体，AhR可以结合并被各种结构上不同的 化合物和这些配体可以产生明显不同的AhR依赖性反应，无论是在幅度 和基因诱导的特异性。基于我们的定点突变和功能分析研究 AhR配体结合结构域（LBD）的同源性模型显示， 这些不同的配体与AhR LBD内的残基，提供了新的见解，如何配体可以激活 AhR。目前认为，AhR依赖性基因表达反应的多样性与不同的细胞因子的表达有关。 配体的差异是由AhR和/或ARNT的结构和功能的配体特异性差异引起的， 差异辅激活因子recuplasty，但这还没有得到证明。虽然经典的AhR机制 包括AhR与ARNT的二聚化以及这种复合物与DNA的结合以调节基因表达， AhR还可以与其他蛋白质（KLF 6和RelB）异源二聚体化，结合到明显不同的DNA序列 并调节其他基因。因此，给定的配体可以差异调节AhR依赖性基因表达， 通过多种机制给予细胞或组织。我们假设AhR复合物的结构可以是 以配体选择性方式改变，导致AhR功能在与 结构上不同的配体，这可以有助于AhR反应的多样性。我们建议研究 不同的AhR结合和结构上不同的配体激活是否改变了AhR的结构和功能 AhR和/或其异源二聚体伴侣（ARNT、KLF 6或RelB），如通过配体和DNA结合测量的， 有限的蛋白水解和基因表达分析，并通过分子对接进行解释。配体选择性 AhR异源二聚体特异性共激活物募集至CYP 1A 1、PAI 2或IL 8基因启动子的差异 人和小鼠肝癌细胞将使用染色质免疫沉淀来确定，并将连接配体- 诱导受体结构的改变，以改变完整细胞中的功能分析。新开发的 AhR：ARNT bHLH-PASA-PASB二聚体的同源性模型将提供一种途径来检查 参与配体依赖性转化/AhR：ARNT二聚化的分子机制以及 用分子动力学方法研究了不同配体对这一过程的影响。本文提出的研究将 提供了详细的分子机制分析，通过这些机制，结构上不同的配体可以 影响AhR及其相关因素，并将深入了解配体依赖性AhR的机制 的转化，配体结构对这些过程的影响和AhR反应的多样性。

The Ah receptor (AhR) is a ligand-dependent transcription factor known to regulate the toxic and biological effects of a variety of exogenous chemicals and these effects result from AhR-dependent gene expression. The AhR is also involved in endogenous developmental and physiological processes, although the responsible endogenous ligand(s) is unknown. While toxic halogenated aromatic hydrocarbons are the prototypical and highest affinity ligands, the AhR can bind and be activated by a diverse range of structurally dissimilar compounds and these ligands can produce distinctly different AhR-dependent responses, both in magnitude and specificity of gene induction. Site-directed mutagenesis and functional analysis studies based on our homology model of the AhR ligand binding domain (LBD) revealed significant differences in the interactions of these diverse ligands with residues within the AhR LBD, providing new insights into how ligands can activate the AhR. It is currently assumed that the diversity in AhR-dependent gene expression response of diverse ligands results from ligand-specific differences in structure and function of the AhR and/or ARNT that leads to differential coactivator recuitment, but this has not been demonstrated. While the classical AhR mechanism involves AhR dimerization with ARNT and binding of this complex to DNA to regulate gene expression, the AhR can also heterodimerize with other proteins (KLF6 and RelB), bind to distinctly different DNA sequences and regulate other genes. Thus, a given ligand can differentially regulate AhR-dependent gene expression in a given cell or tissue by multiple mechanisms. We hypothesize that the structure of the AhR complex can be altered in a ligand-selective manner, resulting in distinct differences in AhR functionality when bound by structurally diverse ligands and this can contribute to the diversity in AhR response. We propose to examine whether differential AhR binding and activation by structurally diverse ligands alters the structure and function of the AhR and/or its heterodimeric partners (ARNT, KLF6 or RelB), as measured by ligand and DNA binding, limited proteolysis and gene expression analysis and interpreted by Molecular Docking. Ligand-selective differences in AhR heterodimer-specific coactivator recruitment to the CYP1A1, PAI2 or IL8 gene promoters in human and mouse hepatoma cells will be determined using chromatin immunoprecipitation and will link ligand- induced alterations in receptor structure to changes in functional analysis in intact cells. The newly developed homology model of the AhR:ARNT bHLH-PASA-PASB dimer will provide an avenue to examine both the molecular mechanisms involved in ligand-dependent transformation/AhR:ARNT dimerization as well as the effect of diverse ligands on this process by Molecular Dynamics methods. The studies proposed here will provide detailed analysis of the molecular mechanisms by which structurally diverse ligands can differentially affect the AhR and its associated factors and will yield insights into the mechanisms of ligand-dependent AhR transformation, the influence of ligand structure on these processes and the diversity of AhR responsiveness.