A Proteomic Analysis of the AHR signaling Network

芳香烃受体(AHR)激动剂，如2，3，7，8-四氯二苯并对二恶英(TCDD)，是一些 人类已知的最有毒的化学物质。他们还占据了EPA-ATSDR前十名中的四名 污染国家优先事项清单的优先物质登记册。这些化合物的毒性是 主要依赖于功能AHR信号复合体的存在。这个建筑群，在 缺少配体包括与90 kDa热休克蛋白(Hsp90)的二聚体结合的AHR， 免疫亲和素样蛋白、ARA9(也称为XAP2和AIP)以及可能的几个其他因素(例如 Pp60src，p21)。这些伴侣的作用和作用机制在很大程度上仍不清楚。 我们最近的初步实验表明，ARA9可能通过招募其他细胞因子来发挥作用 AHR胞浆复合体。这些细胞因子和其他信号系统在形成和发展过程中所起的作用 AHR胞浆复合体(上游事件)的完整性以及这些其他复合体蛋白如何影响 AHR介导的毒性(下游事件)尚未得到彻底研究。这些信号可能会播放 AHR激动剂在组织特异性生物学和毒性中的重要作用。我们的初步数据和最近的 文献已经引导我们假设：上游和下游的二级信号都在 AHR活性的直接影响在AHR介导的信号和毒性中的重要作用 胞浆复合体和下游信号级联的扰动。为了解决这一假设， 该项目将在四个特定的目标(SA)中研究二级信号对AHR生物学的影响。 SA1)鉴定和鉴定在肝脏和免疫细胞中能够与AHR相互作用的蛋白质 串联亲和纯化、质谱学和逆转录病毒检测配体的存在和缺失 介导性基因转移。 Sa2)使用质谱仪确定AHR复合体成员在配体暴露后的命运，并 逆转录病毒介导的基因转移。 SA3)使用RNAi、瞬时RNAi表征AHR相互作用蛋白在配体诱导的信号转导中的作用 转染法和功能分析。 SA4)使用第一个AIMS中确定的蛋白质创建AHR的功能相互作用网络图，并 发布报告，并确定其与监管网络的重叠。 这些目标的完成将创建AHR蛋白质相互作用网络(AHR-PIN)的详细图景 并且该PIN中的蛋白质与AHR介导的毒性的功能后果直接相关。最后， 将开发的计算模型将产生新的机械方向，以理解 AHR配体的毒性，并允许对超级基金站点进行更准确的风险评估。

Aryl hydrocarbon receptor (AHR) agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are some of the most toxic chemicals known to man. They also hold 4 of the top 10 positions within the EPA-ATSDR registry of priority substances that contaminate National Priority List. The toxicity of these compounds is primarily dependent upon the presence of a functional AHR signaling complex. This complex, in the absence of ligand consists of the AHR bound to a dimer of the heat shock protein of 90 kDa (Hsp90), the immunophilin-like protein, ARA9 (also known as XAP2 and AIP) and possibly several other factors (eg pp60src, p21). The role these chaperones play and their mechanism of action remains largely unknown. Our recent preliminary experiments suggest that ARA9 may function by recruiting other cellular factors to the AHR cytosolic complex. The role these cellular factors and other signaling systems play in the formation and integrity of the AHR cytosolic complex (upstream events) and how these other complex proteins influence AHR mediated toxicity (downstream events) has not been thoroughly explored. These signals may play important roles in the tissue specific biology and toxicity of AHR agonists. Our preliminary data and recent literature have led us to hypothesize: Secondary signaling, both upstream and downstream, plays an important role in AHR mediated signaling and toxicity through direct influence of the activity of the AHR cytosolic complex and perturbations of downstream signaling cascades. To address the hypothesis this project will look at the effects of secondary signaling on AHR biology in four specific aims (SA). SA1) Identify and characterize the proteins capable of interacting with the AHR in liver and immune cells in the absence and presence of ligand using tandem affinity purification, mass spectrometry and retroviral mediated gene transfer. SA2) Determine the fate of AHR complex members following ligand exposure using mass spectrometry and retroviral mediated gene transfer. SA3) Characterize the role of AHR-interacting proteins in ligand-induced signaling using RNAi, transient transfections and functional assays. SA4) Create a functional interaction network map for the AHR using proteins identified in the first aims and published reports and determine its overlap with regulatory networks. The completion of these aims will create a detailed picture of the AHR protein interaction network (AHR-PIN) and directly relate the proteins in this PIN to functional consequences for AHR mediated toxicity. Finally, the computational model that will be developed will generate new mechanistic directions for understanding the toxicity of AHR ligands and allow more accurate risk assessment for Superfund sites.