Multiple low-affinity aryl hydrocarbon receptors in the frog Xenopus laevis

非洲爪蟾体内的多个低亲和力芳烃受体

• 批准号: 7902975
• 负责人: WADE H POWELL
• 金额: $ 2.5万
• 依托单位: KENYON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7902975
• 关键词: Accounting; Affinity; African; Agonist; Amino Acid Sequence; Amino Acids; Animals; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Benzo(a)pyrene; Binding; Biological; Biological Assay; Biological Models; CYP1A1 gene; CYP1A6 gene; Carbazoles; Carboxylic Acids; Cells; Chemicals; Data; Development; Dioxins; Dose; Elements; Embryo; Enhancers; Environmental Pollution; Esters; Exhibits; Fishes; Furans; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Homology Modeling; Human; In Situ Hybridization; In Vitro; Individual; Indoles; Ligand Binding; Ligand Binding Domain; Ligands; Luciferases; Measures; Mediating; Mouse Strains; Mus; Mutate; Mutation; Pattern; Phenotype; Phylogenetic Analysis; Physiological; Play; Polychlorinated Biphenyls; Population; Positioning Attribute; Property; Protein Binding; Proteins; Rana; Receptor Signaling; Relative (related person); Reporter Genes; Research; Risk Assessment; Role; Sampling; Scientist; Side; Signal Pathway; Signal Transduction Pathway; Site-Directed Mutagenesis; Small Interfering RNA; Structure; Structure-Activity Relationship; Tadpoles; Testing; Thiazoles; Toxic effect; Toxicity Tests; Toxicology; Variant; Vertebrates; Xenobiotics; Xenopus; Xenopus laevis; Xenopus sp.; activating transcription factor; aryl hydrocarbon receptor ligand; base; carbazole; chlorination; cholanthrene; comparative; environmental chemical; human AHR protein; interest; mouse Ahr protein; novel; paralogous gene; preference; promoter; receptor binding; receptor expression; research study; response; toxicant

DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that mediates the biological and toxicological effects of a broad range of structurally diverse chemicals, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Inherent properties of the AHR signaling pathway, including AHR expression levels and the affinity of AHR for specific ligands, can underlie large variations in the relative potency of different ligands and the sensitivity of different animal groups. However, neither the exact mechanisms by which AHR activity leads to toxicity nor the endogenous, non-toxicological functions of AHR signaling are well understood. Relative to most other vertebrates, frogs are extremely insensitive to TCDD toxicity. Our group has identified two AHRs from the African clawed frog (Xenopus laevis), recently duplicated paralogs called AHR1? and AHR1?. Both proteins bind TCDD with at least 25-fold lower affinity than the AHR from a highly sensitive strain of mouse, likely accounting for the dioxin-insensitive phenotype. The proposed project will take advantage of the unique amino acid sequence, functional properties, and phylogenetic position of the frog AHRs to probe their structural interactions with a range of xenobiotic and naturally occurring ligands and to contrast their function with AHRs from TCDD-sensitive species and with each other. We propose three specific aims: (1) Using site-directed mutagenesis to make the frog AHRs more "mouse-like," we will test the hypothesis that changes in one or a few amino acids within the putative ligand binding domain confer low TCDD affinity. This comparative approach will contribute significantly to the identification of important structural features of AHR's ligand binding pocket. (2) We will determine the relative potency of structurally diverse candidate ligands. Although X. laevis AHRs bind TCDD with low affinity, they may remain highly responsive to structurally distinct compounds, especially putative endogenous ligands. We will test this hypothesis by establishing structure-activity relationships for a range of candidate ligands, including indole-containing compounds that bind mammalian AHRs. (3) We will determine the functional differences between AHR1? and AHR1?, examining expression patterns, enhancer preferences, and broad-based changes in gene expression mediated by individual paralogs. These studies will test the hypothesis that the AHR paralogs exhibit distinct functions, possibly partitioning multiple roles of the single mammalian AHR. Overall, this comparative approach in a novel model system will provide important basic information about the structure and function of all vertebrate AHRs. Understanding the differences between frog and human AHRs will also aid risk assessment by refining interpretation of toxicological data derived from FETAX (Frog Embryo Teratogenesis Assay-Xenopus) and similar developmental toxicity tests that employ frog embryos. This project will study the aryl hydrocarbon receptor (AHR), a protein that mediates the toxic effects of environmental contaminants such as dioxin. We will compare the structure and function of the AHR from frogs, which are insensitive to dioxin toxicity, with AHRs from more sensitive animals like mice and humans. This comparative research will help scientists understand important differences between frogs and humans as they interpret toxicology studies using frog embryos as a model system for measuring the effects of chemicals and environmental samples. It will also provide basic information about the original, non-toxicological function of the AHR.

描述（由申请人提供）：芳烃受体（AHR）是一种配体激活的转录因子，可介导多种结构多样的化学品（包括2，3，7，8-四氯二苯并-对-二恶英（TCDD））的生物学和毒理学效应。AHR信号传导途径的固有特性，包括AHR表达水平和AHR对特定配体的亲和力，可以成为不同配体的相对效力和不同动物组的敏感性的巨大变化的基础。然而，无论是AHR活性导致毒性的确切机制，还是AHR信号传导的内源性、非毒理学功能，都没有很好地理解。相对于大多数其他脊椎动物，青蛙对四氯二苯并对二恶英毒性极不敏感。我们的小组已经确定了两个AHR从非洲爪蟾（非洲爪蟾），最近复制的旁系同源称为AHR 1？AHR1？这两种蛋白质结合TCDD的亲和力比来自高度敏感的小鼠品系的AHR低至少25倍，这可能是二恶英不敏感表型的原因。拟议的项目将利用独特的氨基酸序列，功能特性，和系统发育的青蛙AHRs的位置来探测它们的结构与一系列异生物质和天然存在的配体的相互作用，并将它们的功能与TCDD敏感物种的AHRs进行对比。我们提出了三个具体的目标：（1）使用定点突变，使青蛙AHRs更“小鼠样”，我们将测试的假设，在一个或几个氨基酸的变化，在假定的配体结合域赋予低TCDD亲和力。这种比较的方法将有助于显着的AHR的配体结合口袋的重要结构特征的识别。(2)我们将确定不同结构的候选配体的相对效力。虽然X.虽然非洲乳杆菌AHR以低亲和力结合TCDD，但它们可对结构上不同的化合物，特别是推定的内源性配体保持高度响应。我们将通过建立一系列候选配体（包括结合哺乳动物AHR的含吲哚化合物）的结构-活性关系来测试这一假设。(3)我们将确定AHR 1？AHR1？，检查表达模式、增强子偏好和由个体旁系同源物介导的基因表达的广泛变化。这些研究将测试的假设，即AHR旁系同源物表现出不同的功能，可能分区的单一哺乳动物AHR的多个角色。总的来说，这种比较方法在一个新的模型系统将提供重要的基本信息的结构和功能的所有脊椎动物AHR。了解青蛙和人类AHRs之间的差异也将有助于风险评估，通过完善来自FETAX（青蛙胚胎致畸试验-非洲爪蟾）和类似的发育毒性试验，采用青蛙胚胎的毒理学数据的解释。该项目将研究芳香烃受体（AHR），这是一种介导二恶英等环境污染物毒性作用的蛋白质。我们将比较来自青蛙的AHR的结构和功能，青蛙对二恶英毒性不敏感，来自更敏感的动物如小鼠和人类的AHR。这项比较研究将帮助科学家了解青蛙和人类之间的重要差异，因为他们解释了使用青蛙胚胎作为测量化学品和环境样本影响的模型系统的毒理学研究。它还将提供有关AHR的原始非毒理学功能的基本信息。