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）：芳烃受体（AHR）介导多种结构配体的毒理学效应，包括2,3,7,8四氯二苯并-对二恶英（TCDD）。AHR的性质，包括它的配体结合亲和力，可能是不同激动剂的相对效力和不同动物群体敏感性的广泛差异的基础。青蛙对TCDD的毒性非常不敏感，非洲爪蟾的AHR与TCDD的亲和力比一种高度敏感的小鼠菌株的AHR低20倍。除了在外源毒性中发挥作用外，AHR还可能介导天然配体的作用。一个候选内源性配体是6-甲酰基林多洛[3,2-b]咔唑（FICZ），一种色氨酸光产物。在之前的资助期内，我们发现FICZ在蛙细胞系中是一种有效的AHR激动剂，其诱导细胞色素P4501A （CYP1A）的效力至少是TCDD的30倍。这表明FICZ与青蛙AHRs结合的亲和力高于TCDD。在培养中，FICZ的效力随着时间的推移而下降，这表明与TCDD不同，它是由其诱导的酶代谢的。这两个特性与假设一致，即尽管青蛙ahr已经失去了高亲和力结合TCDD的能力，但它们仍然对一种假定的生理配体表现出高反应性。在本区域拨款续期申请中，我们提出了一套完整的实验，逻辑上遵循我们之前对青蛙AHR信号的描述。该项目将结合TCDD和FICZ探索青蛙AHR的结构和作用，有三个具体目标：(1)在最近开发的X. laevis AHR配体结合域的同源性模型的指导下，我们将使用位点定向诱变使青蛙AHR更“像老鼠”，确定哪些差异导致低TCDD亲和力。我们还将检验FICZ结合的决定因素与TCDD的决定因素不同的假设。这些研究将确定AHR的配体结合袋与天然和异种配体的重要结构特征。(2)利用RNA-seq，我们将检验FICZ和TCDD引起不同靶基因组表达变化的假设。这种高度敏感和定量的方法将改进Affymetrix微阵列的初步研究，使商业阵列上的许多未知转录物得以识别，填补了非洲爪蟾基因组学中巨大的注释空白。(3)我们将确定CYP1A6和CYP1A7的催化特异性，这是进化上独特的CYP1A亲缘关系。这些研究将验证这样一个假设，即每种酶催化结构域的关键氨基酸差异决定了底物偏好，包括对FICZ代谢的特异性。总的来说，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的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，还将有助于风险评估，因为它可以对FETAX和使用青蛙胚胎的类似发育毒性试验得出的毒理学数据进行更准确的解释。