Understanding the origins and mechanisms of aryl hydrocarbon receptor promiscuity

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 are performing 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.

The aryl hydrocarbon receptor (AHR) mediates the toxicity of dioxin-like environmental contaminants and has been implicated in the etiology and progression of inflammatory diseases and cancer, but a comprehensive, mechanistic understanding of how chemicals cause toxicity via the AHR has been hindered by the complexity of its physiological functions and the diversity of its ligands (AHR promiscuity). The proposed research will reveal the properties of the primordial ligand-activated AHR and the molecular mechanisms underlying the evolution of promiscuity. This basic research will provide fundamental new insights into the natural physiological ligands and biological functions of the AHR, reveal the genetic and structural mechanisms underlying AHR ligand recognition, and elucidate how and why AHR function is disrupted by exposure to anthropogenic environmental contaminants.

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