Understanding the origins and mechanisms of aryl hydrocarbon receptor promiscuity

项目摘要/摘要 芳香烃受体(AHR)是一种在脊椎动物中由配体激活的bHLH-PAS蛋白。 转录因子，在外源代谢酶的调节中起重要作用 多种环境污染物的毒性机制，包括氯化二恶英，如 2，3，7，8-四氯二苯并-对二恶英(TCDD)、一些多氯联苯(PCbs)、多核芳烃 碳氢化合物(PAHs)和一些新出现的污染物。AHR还参与了各种 生理过程包括发育、造血、免疫、宿主-微生物组相互作用和 屏障器官功能。与大多数配体激活的转录因子具有窄配体相反- 特异性，脊椎动物的AHR是高度混杂的，识别不同的化学物质。除了……之外 众所周知的外源生物，AHR配体包括饮食中的植物化学物质，微生物群衍生的微生物 代谢物和内源性代谢物，如色氨酸分解代谢物，所有这些共同有助于 内部化学曝光体。从机制上全面理解AHR在应对 环境暴露由于其复杂的生理功能和 其配体的多样性令人眼花缭乱。在这里，我们提出了一套创新的分子研究来阐明 AHR配体依赖的序列-结构-功能决定因素及其配体多样性的起源。 有证据表明，AHR是从一个不依赖配体(构成活性)的祖先进化而来的。这个 拟议的基础研究将在实验上确定进化轨迹和潜在的遗传和 驱动AHR配体依赖和混杂进化的结构机制。在目标1中，我们将 通过系统的实验建立现代物种AHRs的配基特异性 后生动物AHR系统发育多样性的评估，包括新的无脊椎动物和早期脊椎动物 AHRS。在目标2中，我们将使用祖先序列重建(ASR)来复活祖先AHR蛋白 然后确定它们与配体结合的敏感性和特异性，揭示祖先和 衍生配体。在目标3中，我们将使用系统发育和蛋白质结构分析来确定候选 历史上的氨基酸变化导致了配基结合的获得和混杂的进化。我们 将通过设计包含这些替换的祖先和现存蛋白质来检验这些假设 试验性地评估它们的功能。了解原始配体的祖先属性- 激活的AHR和推动滥交演变的机制将提供基本的新见解 深入研究现有AHR的天然生理配体和生物学功能，揭示其遗传和结构 AHR配体识别的潜在机制，并阐明AHR功能如何以及为什么被 人为环境污染物。

Project Summary/Abstract The aryl hydrocarbon receptor (AHR) is a bHLH-PAS protein that in vertebrate animals is a ligand-activated transcription factor that plays essential roles in the regulation of xenobiotic-metabolizing enzymes and in the mechanisms of toxicity of numerous environmental contaminants, including chlorinated dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), some polychlorinated biphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs), and some emerging contaminants. The AHR is also involved in a variety of physiological processes including development, hematopoiesis, immunity, host-microbiome interactions, and barrier organ function. In contrast to most ligand-activated transcription factors that have narrow ligand- specificity, the vertebrate AHR is highly promiscuous, recognizing a diverse array of chemicals. In addition to the well-known xenobiotics, AHR ligands include dietary phytochemicals, microbiome-derived microbial metabolites, and endogenous metabolites such as tryptophan catabolites, all of which collectively contribute to the internal chemical exposome. A comprehensive mechanistic understanding of AHR’s role in the response to environmental exposures has been hindered by the complexity of its physiological functions and the bewildering diversity of its ligands. Here, we propose a set of innovative molecular studies to elucidate the sequence-structure-function determinants of AHR ligand-dependence and the origin of its ligand diversity. Evidence suggests that the AHR evolved from a ligand-independent (constitutively active) ancestor. The proposed basic research will experimentally determine the evolutionary trajectory and underlying genetic and structural mechanisms that drove the evolution of AHR ligand-dependence and promiscuity. In Aim 1, we will establish the ligand-specificity of AHRs from present-day species through a systematic experimental assessment of phylogenetically diverse metazoan AHRs, including new invertebrate and early vertebrate AHRs. In Aim 2, we will use ancestral sequence reconstruction (ASR) to “resurrect” ancestral AHR proteins and then determine their ligand-binding sensitivity and specificity, revealing the identities of ancestral and derived ligands. In Aim 3, we will use phylogenetic and protein structural analysis to identify candidate historical amino acid changes that caused the acquisition of ligand-binding and evolution of promiscuity. We will test these hypotheses by engineering ancestral and extant proteins containing these substitutions and experimentally assessing their function. Understanding the ancestral properties of the primordial ligand- activated AHR and the mechanisms that drove the evolution of promiscuity will provide essential new insights into the natural physiological ligands and biological functions of extant AHR, reveal the genetic and structural mechanisms underlying AHR ligand recognition, and elucidate how and why AHR function is disrupted by anthropogenic environmental contaminants.