Year: 2010
Type: dissertation
Abstract: The Aryl-hydrocarbon Receptor (AhR) is a basic Helix-Loop-Helix Per-ARNT-Sim (bHLH.PAS) transcription factor (TF) which binds partner protein Aryl hydrocarbon Receptor Nuclear Translocator (ARNT), in order to activate target genes in response to environmental or endogenous stimuli. The PAS region of these TFs consists of two adjacent repeats of the PAS domain, where the PAS repeat defines dimerization specificity and also serves as a primary sensor in exogenous ligand activation of AhR. Active AhR/ARNT heterodimer binds specific DNA sequences, termed Xenobiotic Response Elements (XRE). The molecular detail of interactions that dictate dimerization and DNA binding specificity are unknown for this TF family. In addition, active AhR has recently been shown to function as a recognition component of an E3 ubiquitin ligase. Reports that AhR null mice have poor fertility and defects in liver vasculature are indicative of the potential for a number of endogenous roles. Research into activation of AhR has highlighted that post-translational modifications may affect function by regulating subcellular localization. The complex regulatory outcomes of AhR expression and activation require a number of approaches to elucidate mechanistic information. In this thesis, a structural investigation of heterodimerization and DNA binding has been used to propose a molecular mechanism for target gene recognition and activation following XRE binding. Crystallographic approaches have yielded crystals of bHLH.PAS-A regions of AhR/ARNT heterodimer bound to DNA. Atomic force microscopy and small angle x-ray scattering analyses have illustrated an XRE binding mechanism whereby the DNA is bent, and the PASA region of the dimer flattens around the DNA. A targeted mutagenesis screen of the AhR ligand binding domain (LBD) was performed to investigate polycyclic aromatic hydrocarbon (PAH) and atypical ligand binding specificity. In parallel, mutant AhR proteins were assessed for inducibility by nonexogenous ligand modes of activation, including cell suspension and application of shear stressed serum. This process identified an LBD mutant selectively activated by novel ligand YH439, and completely inactive following PAH, cell suspension and shear stressed serum treatments, inferring the potential for differential ligand binding pocket access by YH439. Finally, given the complex output following expression and activation of AhR, regulation by post-translational modification was investigated as a potential means of subtle regulation of signalling fate. A thorough analysis of untreated AhR has revealed a concert of modifications occurring on functionally relevant regions of the protein that are implicated in regulating subcellular localization, protein: protein interactions, and potentially, protein stability. Preliminary analyses of YH439 and cell suspension treated AhR has additionally indicated the possibility of activation state specific modification patterns. In summary, this thesis describes: Novel approaches to structural characterisation of a bHLH.PAS protein dimer bound to DNA; atypical ligand binding to a novel site of AhR; and an analysis of a proposed AhR PTM code.
Author: Fiona Whelan
Language: English
Subfield: Health, Toxicology and Mutagenesis
Field: Environmental Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2758852219