Low-affinity aryl hydrocarbon receptors in the frog Xenopus laevis

描述(申请人提供)：芳烃受体(AHR)介导结构多样的配体的毒理效应，包括2，3，7，8四氯二苯并对二恶英(TCDD)。AHR的性质，包括其与配体的结合亲和力，可以导致不同激动剂的相对效力和不同动物群体的敏感性的巨大差异。青蛙对TCDD毒性极其不敏感，非洲爪蛙(非洲爪蛙)的AHR与TCDD的亲和力比高度敏感的小鼠品系的AHR低20倍。除了在异种生物毒性中的作用外，AHR还可能介导天然配体的作用。一个候选的内源性配体是6-甲酰吲哚[3，2-b]咔唑(FICZ)，一种色氨酸光产物。在之前的授权期，我们证明了FICZ是一种在青蛙细胞系中有效的AHR激动剂--在诱导细胞色素P4501A(CyP1A)方面至少比TCDD强30倍。这表明FICZ与蛙AHR结合的亲和力高于TCDD。FICZ的效力随着培养时间的延长而下降，这表明与TCDD不同的是，它是由它诱导的酶代谢的。这两个特性与青蛙AHR失去了与TCDD高亲和力结合的能力的假设是一致的，但它们仍然表现出对假定的生理配体的高反应性。在这方面，我们提出了一套完整的实验，这些实验符合我们之前对青蛙AHR信令的描述。本项目将结合TCDD和FICZ来探索青蛙AHR的结构和作用，承担三个具体目标：(1)在最近开发的莱维氏X.laevis AHR配体结合结构域的同源模型的指导下，我们将使用定点突变来使青蛙AHR更像“老鼠”，确定哪些差异导致TCDD亲和力较低。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与天然和异种配体结合口袋的重要结构特征。(2)使用RNA-seq，我们将检验FICZ和TCDD诱导不同靶基因集表达变化的假设。这种高度敏感和定量的方法将改进Affymetrix微阵列的初步研究，使其能够识别商业阵列上的许多未知转录本，填补非洲爪哇基因组学中巨大的注释空白。(3)我们将确定CYP1A6和CYP1A7这两个进化上独特的细胞色素P1a类似物的催化专一性。这些研究将检验这样一种假设，即每种酶催化区域的关键氨基酸差异会带来底物偏好，包括FICZ代谢的特异性。总体而言，我们的比较方法将青蛙AHR信号通路的独特功能视为“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之间的差异也将有助于通过完善对FETAX和使用青蛙胚胎进行的类似发育毒性测试得出的毒理学数据的解释来进行风险评估。 与公共卫生相关：该项目将研究芳香烃受体(AHR)，这是一种介导环境污染物(如二恶英)毒性效应的蛋白质。这种蛋白质在我们试图更好地了解的生理和发育中也有重要的作用。我们将比较对二恶英毒性不敏感的青蛙AHR的结构、功能和下游效应，以及来自更敏感动物(如老鼠和人类)的AHR。这项比较研究将帮助科学家确定这一系统中所有脊椎动物共有的最重要的特征。它还将有助于理解青蛙和人类之间的重要差异，因为他们使用青蛙胚胎作为衡量化学品和环境样本影响的模型系统来解释毒理学研究。

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.