A Proteomic Analysis of the AHR signaling Network

AHR 信号网络的蛋白质组学分析

• 批准号: 8055595
• 负责人: JOHN J LAPRES
• 金额: $ 25.88万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055595
• 关键词: ARA9 protein; Address; Affinity Chromatography; Agonist; Arts; Aryl Hydrocarbon Receptor; Biological Assay; Biological Process; Biology; Cells; Complex; Computer Simulation; Cytosol; Data; Dioxins; Event; Exposure to; Gene Transfer; Genes; Goals; Heat-Shock Proteins 90; Immune; Immunophilins; In Vitro; Ligands; Literature; Liver; Location; Maps; Mass Spectrum Analysis; Mediating; Modeling; Molecular Chaperones; Molecular Conformation; Nature; Play; Poison; Positioning Attribute; Protein Analysis; Proteins; Proteomics; Publishing; RNA Interference; Receptor Signaling; Recruitment Activity; Registries; Reporting; Risk Assessment; Role; Series; Signal Transduction; Site; Small Interfering RNA; System; Testing; Tetrachlorodibenzodioxin; Time; Tissues; Toxic effect; Transfection; Viral Vector; Western Blotting; aryl hydrocarbon receptor ligand; dimer; in vitro Assay; knock-down; man; member; overexpression; protein complex; receptor; receptor binding; receptor-mediated signaling; research study; retroviral-mediated; superfund site; tool

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.

芳香烃受体（AHR）激动剂，例如2，3，7，8-四氯二苯并-对-二恶英（TCDD），是一些 人类所知的最有毒的化学物质。在EPA-ATSDR中，它们也占据了前10名中的4名 污染国家优先清单的优先物质登记册。这些化合物的毒性是 主要依赖于功能性AHR信号复合物的存在。这个复杂的，在 配体的缺乏由AHR与90 kDa的热休克蛋白（Hsp 90）的二聚体结合组成， 免疫亲素样蛋白ARA 9（也称为XAP 2和AIP）和可能的几种其他因子（例如 pp60src，p21）。这些分子伴侣所起的作用及其作用机制在很大程度上仍然未知。 我们最近的初步实验表明，ARA 9可能通过招募其他细胞因子来发挥作用。 AHR胞质复合物。这些细胞因子和其他信号系统在形成和 AHR胞质复合物的完整性（上游事件）以及这些其他复合蛋白如何影响 AHR介导的毒性（下游事件）尚未得到彻底研究。这些信号可能发挥作用 在AHR激动剂的组织特异性生物学和毒性中起重要作用。我们的初步数据和最近的 文献使我们假设：次级信号，上游和下游， 通过直接影响AHR的活性，在AHR介导的信号传导和毒性中发挥重要作用 胞质复合物和下游信号级联的扰动。为了解决这个假设， 该项目将着眼于次级信号对AHR生物学在四个特定目标（SA）的影响。 SA 1）鉴定和表征能够与肝脏和免疫细胞中的AHR相互作用的蛋白质， 使用串联亲和纯化、质谱和逆转录病毒 介导的基因转移 SA 2）使用质谱法测定配体暴露后AHR复合物成员的命运，以及 逆转录病毒介导的基因转移。 SA 3）使用RNAi、瞬时RNAi、瞬时RNAi、瞬时RNAi和瞬时RNAi来表征AHR相互作用蛋白在配体诱导的信号传导中的作用。 转染和功能测定。 SA 4）使用第一个目标中鉴定的蛋白质创建AHR的功能相互作用网络图，以及 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）的详细图片 并将PIN中的蛋白质与AHR介导的毒性的功能结果直接相关。最后 将开发的计算模型将产生新的机械方向， AHR配体的毒性，并允许更准确的风险评估超级基金网站。