Low-affinity aryl hydrocarbon receptors in the frog Xenopus laevis

非洲爪蟾中的低亲和力芳烃受体

• 批准号: 8035190
• 负责人: WADE H POWELL
• 金额: $ 28.68万
• 依托单位: KENYON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2014-09-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035190
• 关键词: Low; affinity; aryl; hydrocarbon; receptors

DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) mediates the toxicological effects of structurally diverse ligands, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Properties of the AHR, including its ligand binding affinity, can underlie wide variations in the relative potency of different agonists and the sensitivity of different animal groups. Frogs are extremely insensitive to TCDD toxicity, and AHRs from Xenopus laevis (African clawed frog) bind TCDD with >20-fold lower affinity than the AHR from a highly sensitive mouse strain. In addition to its role in xenobiotic toxicity, the AHR may also mediate the effects of natural ligands. One candidate endogenous ligand is 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct. In the previous grant period, we showed FICZ is a potent AHR agonist in a frog cell line--at least 30-fold more potent than TCDD for cytochrome P4501A (CYP1A) induction. This suggests that FICZ binds frog AHRs with higher affinity than TCDD. FICZ potency declined with time in culture, suggesting that unlike TCDD, it is metabolized by enzymes it induces. These two properties are consistent with the hypothesis that although frog AHRs have lost the ability to bind TCDD with high affinity, they nonetheless exhibit high responsiveness to a putative physiological ligand. In this AREA grant renewal application, we propose an integrated set of experiments that logically follow our previous characterizations of frog AHR signaling. This project will probe the structure and effects of frog AHRs in conjunction with both TCDD and FICZ, undertaking three specific aims: (1) Guided by a recently developed homology model of the X. laevis AHR ligand binding domains, we will use site-directed mutagenesis to make the frog AHRs more "mouse-like," determining which differences confer low TCDD affinity. We will also test the hypothesis that determinants of FICZ binding differ from those of TCDD. These studies will identify important structural features of AHR's ligand binding pocket with both natural and xenobiotic ligands. (2) Using RNA-seq, we will test the hypothesis that FICZ and TCDD elicit expression changes in distinct sets of target genes. This highly sensitive and quantitative approach will improve on preliminary Affymetrix microarray studies by enabling the identification of the many unknown transcripts on commercial arrays, filling an enormous annotation gap in Xenopus genomics. (3) We will determine the catalytic specificity of CYP1A6 and CYP1A7, evolutionarily unique CYP1A paralogs. These studies will test the hypothesis that key amino acid differences in the catalytic domains of each enzyme confer substrate preferences, including specificity for FICZ metabolism. Overall, our comparative approach treats the unique features of the frog AHR signaling pathway as "mutant phenotypes" to glean important general information about the toxicological and biological functions of this system in humans and other vertebrates. Understanding the differences between frog and human AHRs will also aid risk assessment by refining interpretation of toxicological data derived from FETAX and similar developmental toxicity tests employing frog embryos. PUBLIC HEALTH RELEVANCE: This project will study the aryl hydrocarbon receptor (AHR), a protein that mediates the toxic effects of environmental contaminants such as dioxin. This protein also has important roles in physiology and development which we seek to better understand. We will compare the structure, function, and downstream effects 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 identify the most important features of this system that are shared between all vertebrates. It will also aid in the understanding of 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.

描述（由申请人提供）：芳基烃受体（AHR）介导结构上多样的配体的毒理学作用，包括2,3,7,8 tetrachlorodibenzo-p-Dioxin（TCDD）。 AHR的性质，包括其配体结合亲和力，可以是不同激动剂的相对效力和不同动物群的敏感性的广泛变化。青蛙对TCDD毒性非常不敏感，而来自Xenopus laevis（非洲爪青蛙）的AHR与高度敏感的小鼠菌株的AHR相比，与AHR低20倍。除了其在异生物毒性中的作用外，AHR还可以介导天然配体的作用。一个候选内源配体是色氨酸光生的6型甲基迪洛洛[3,2-b] carbazole（FICZ）。在上一个赠款期间，我们显示FICZ是青蛙细胞系中有效的AHR激动剂 - 对于细胞色素P4501A（CYP1A）诱导，TCDD的效力至少比TCDD高30倍。这表明FICZ结合了比TCDD更高的亲和力的青蛙AHR。 FICZ效力随着文化的时间而下降，表明与TCDD不同，它是由其诱导的酶代谢的。这两种特性与以下假设一致：尽管青蛙AHR失去了与高亲和力结合TCDD的能力，但它们仍然对假定的生理配体具有很高的响应能力。在此领域授予续订应用中，我们提出了一组集成的实验集，从逻辑上讲，我们先前对青蛙AHR信号的表征。该项目将与TCDD和FICZ一起探测青蛙AHR的结构和影响，实现三个具体目标：（1）在X. laevis AHR配体结合域的最近开发的同源性模型的指导下，我们将使用以站点为导向的位置定向。诱变使青蛙AHR更类似于“小鼠”，确定哪些差异赋予低TCDD亲和力。我们还将检验以下假设：FICZ结合的决定因素与TCDD的结合不同。这些研究将确定具有天然和异种配体的AHR配体结合口袋的重要结构特征。 （2）使用RNA-Seq，我们将测试以下假设：FICZ和TCDD在不同的靶基因集中引起表达变化。这种高度敏感和定量的方法将通过在商业阵列上鉴定许多未知的转录本，从而填补Xenopus Genomics中的巨大注释差距，从而改善初步的Affymetrix微阵列研究。 （3）我们将确定CYP1A6和CYP1A7的催化特异性，即进化独特的CYP1A旁系同源物。这些研究将检验以下假设：每种酶会议的催化结构域的关键氨基酸差异赋予底物偏好，包括FICZ代谢的特异性。总体而言，我们的比较方法将青蛙AHR信号通路的独特特征视为“突变表型”，以收集有关该系统在人类和其他脊椎动物中该系统毒理学和生物学功能的重要一般信息。了解青蛙和人类AHR之间的差异还将通过完善对胎儿的毒理学数据的解释以及采用青蛙胚胎的类似发育毒性测试的解释。 公共卫生相关性：该项目将研究芳基烃受体（AHR），该蛋白质介导了二恶英等环境污染物的毒性作用。该蛋白质在生理和发育中也具有重要的作用，我们试图更好地理解。我们将比较青蛙的AHR的结构，功能和下游效应，对二恶英毒性不敏感，以及来自小鼠和人类（如小鼠和人类）的AHR。这项比较研究将帮助科学家确定所有脊椎动物之间共享该系统的最重要特征。它还将有助于理解青蛙和人类之间的重要差异，因为它们使用青蛙胚胎作为测量化学物质和环境样品影响的模型系统来解释毒理学研究。