Investigating aryl hydrocarbon receptor-cofactor interactions that mediate toxicity

研究介导毒性的芳烃受体-辅因子相互作用

• 批准号: 9024795
• 负责人: Daniel Gorelick
• 金额: $ 52.06万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9024795
• 关键词: 3-Methylcholanthrene; ARNT gene; ARNT protein; Acute; Address; Adverse effects; Affect; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Benzo(a)pyrene; Binding; Biochemical; Cardiac Edema; Cardiotoxicity; Cardiovascular Physiology; Cardiovascular system; Chemicals; Co-Immunoprecipitations; Complement; Complex; DNA; Data; Development; Dioxins; Disease; Embryo; Embryonic Heart; Epitopes; Estrogen Receptor alpha; Estrogen Receptors; Exposure to; Family; Foundations; Gene Expression; Genes; Genetic; Goals; Health; Heart; Heart Abnormalities; Human; Immune system; Knock-in; Knowledge; Lead; Ligand Binding; Ligands; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Mutagenesis; Organ; Pathology; Phenotype; Proteins; Proteomics; Receptor Signaling; Recruitment Activity; Regulation; Role; Signal Pathway; Structure; Testing; Tetrachlorodibenzodioxin; Tissue-Specific Gene Expression; Tissues; Toxic effect; Zebrafish; aryl hydrocarbon receptor ligand; aryl hydrocarbons; cofactor; genetic approach; improved; in vivo; pollutant; public health relevance; receptor; research study; response; transcription factor; tumor growth

 DESCRIPTION (provided by applicant): Exposure to byproducts of industrial combustion, such as dioxins and polycyclic aromatic hydrocarbons (PAH), causes multi-organ phenotypes including cardiotoxicity. Understanding the signaling pathways by which such compounds cause toxicity is crucial for predicting acceptable exposure levels and for reversing the effects of adverse exposure. Dioxins and PAHs activate aryl hydrocarbon receptors (AHR), ligand-dependent transcription factors that recruit cofactors to DNA and directly regulate gene expression. However, our knowledge of AHR cofactor recruitment and regulation is limited. The goal of this application is to identify the transcriptional cofactors by which AHR mediates cardiotoxicity and test the hypothesis that AHR ligands modulate cofactor recruitment. Structurally distinct AHR ligands induce differential transcriptional responses, suggesting that different ligands bind AHR and recruit different cofactors, leading to differential gene expression Consistent with this hypothesis, our preliminary data in zebrafish embryos, an established model of dioxin and PAH toxicity, indicates that AHR-mediated cardiotoxicity is ligand dependent. Acute exposure to the AHR ligand 2, 3, 7, 8-tetrachlorodibenzodioxin (TCDD), but not the AHR ligands 3-methylcholanthrene (3MC) or benzo (a) pyrene (BaP), caused cardiac edema and heart malformation. While the idea of ligand-dependent cofactor recruitment has been established for the estrogen receptor family of transcription factors, whether AHRs act in a similar manner is less well understood. Approximately 20 AHR cofactors have been identified, yet aside from the ARNT protein, it is not known which cofactors are required for cardiotoxicity or whether cofactors are recruited in a ligand-dependent manner. We propose that TCDD-AHR recruits a different complex of transcriptional cofactors than either 3MC-AHR or BaP-AHR and that differential cofactor recruitment underlies ligand-dependent gene expression and cardiotoxicity. In Aim 1 we will identify AHR cofactors required for TCDD-dependent cardiotoxicity. Using genetic approaches, we will test whether candidate cofactors are required for TCDD cardiotoxicity. We will also use unbiased biochemical and proteomic approaches to purify TCDD-AHR from the developing heart and identify interacting proteins. In Aim 2 we will identify ligand-dependent AHR cofactors from whole embryos and select tissues. Using zebrafish `knock-in' strains with epitope tags inserted into endogenous AHR signaling genes, we will test whether known cofactors differentially interact with endogenous AHR depending on the AHR ligand. We will also purify AHR from zebrafish embryos and use mass spectrometry to identify cofactors bound to AHR following exposure to TCDD, 3MC or BaP. Results from this application will identify a mechanism of TCDD cardiotoxicity and will provide a foundation for elucidating how the structure of AHR changes in response to differential ligand binding. Furthermore, our results will increase our understanding of the role of cofactors in endogenous AHR signaling, which may lead to improved treatment for diseases where AHR signaling is abnormal.

 描述（由申请人提供）：接触工业燃烧的副产品，如二恶英和多环芳烃 (PAH)，会导致多器官表型，包括心脏毒性。了解此类化合物引起毒性的信号传导途径对于预测可接受的接触水平至关重要。并逆转不良接触的影响，激活芳基碳氢化合物受体 (AHR)，这是一种配体依赖性转录因子，可将辅助因子招募到 DNA 中并直接调节基因。然而，我们对 AHR 辅因子募集和调节的了解有限，该应用的目的是鉴定 AHR 介导心脏毒性的转录辅因子，并测试 AHR 配体调节结构不同的 AHR 配体诱导差异转录反应的假设。 ，表明不同的配体结合AHR并招募不同的辅助因子，导致差异基因表达与这一假设相一致，我们在斑马鱼胚胎中的初步数据，二恶英和二恶英的建立模型PAH 毒性表明 AHR 介导的心脏毒性是配体依赖性的，急性暴露于 AHR 配体 2, 3, 7, 8-四氯二苯并二恶英 (TCDD)，而不是 AHR 配体 3-甲基胆蒽 (3MC) 或苯并 (a) 芘（ BaP），引起心脏水肿和心脏畸形，而对于转录因子的雌激素受体家族，配体依赖性辅助因子募集的想法已经建立。 AHR 以类似的方式发挥作用尚不清楚。已鉴定出大约 20 种 AHR 辅因子，但除 ARNT 蛋白外，尚不清楚哪些辅因子是心脏毒性所必需的，也不知道辅因子是否以配体依赖性方式招募。 TCDD-AHR 募集了与 3MC-AHR 或 BaP-AHR 不同的转录辅因子复合物，并且差异辅因子募集是配体依赖性基因表达和毒性心脏的基础。在目标 1 中，我们将使用遗传方法鉴定 TCDD 依赖性心脏毒性所需的 AHR 辅因子，我们还将使用公正的生化和蛋白质组学方法从发育中的心脏和心脏中纯化 TCDD-AHR。在目标 2 中，我们将使用斑马鱼“敲入”品系从整个胚胎和选择的组织中鉴定配体依赖性 AHR 辅因子。将表位标签插入内源性 AHR 信号基因中，我们将测试已知的辅助因子是否根据 AHR 配体与内源性 AHR 存在差异相互作用。我们还将从斑马鱼胚胎中纯化 AHR，并使用质谱法来鉴定暴露于 TCDD、3MC 后与 AHR 结合的辅助因子。该应用的结果将确定 TCDD 心脏毒性的机制，并将为阐明 AHR 的结构如何响应差异配体而变化提供基础。此外，我们的结果将增加我们对辅助因子在内源性 AHR 信号传导中的作用的理解，这可能会改善 AHR 信号传导异常疾病的治疗。