The dioxin receptor (DR), a transcription factor with basic-helix-loop-helix/PERARNTSIM (bHLH/PAS) homology domains, is activated by toxic xenobiotic ligands leading to severe physiological disturbances most of which are due to deregulation of receptor’s central role in normal development. Activation mechanisms of DR in the presence of exogenous or endogenous ligands are poorly understood. Elucidation of factors involved in the activation of the receptor would assist not only in development of an optimal measure for risk assessment of levels of common environmental pollutants but also in providing novel targets for therapeutic interventions. Posttranslational modifications (PTMs) play an indispensable role in all major signal transduction pathways by increasing the inventory of chemical modifications beyond those already present in the side-chains of common amino acids. Thus, by simple on/off or complex patterns generated by these PTMs, they control a myriad of different biological outcomes. Numerous studies that have suggested an important role of posttranslational modifications in DR activation has prompted a search in this direction, however, apart from phosphorylations at Ser36 and Ser68 no other PTM sites are known. Advanced mass spectrometry (MS)-based characterisation of PTMs is an established technique that can comprehensively provide an accurate cast of all PTM variants and their locations on a protein. This thesis reports the first MS-based comprehensive characterisation of all PTM sites of the purified latent DR and preliminary analysis of identified PTM sites of the activated DR in response to developmental signals (suspension-activated DR) and signals leading to toxic outcomes (ligand-activated DR). The PTM map of the latent DR revealed from this study comprises of 25 phosphorylations, 4 monomethyl-lysines, 2 dimethyl-lysines, 1 O-acetyl-serine and 2 O-sulfono-serines. Most of the phosphorylations and other PTMs were present in the conserved regions of the protein. Investigation of the activated samples of the receptor revealed loss of the above repertoire of modifications and possible presence of some rarer modifications such as O-acetyl-serines in suspension-activated instead of O-sulfonations and pyrophosphorylation at Ser716 in both suspension- as well as ligand-activated DR. A comprehensive mutagenesis study is in progress to understand the functional consequence of each of these modification sites and unravel the functional posttranslational system in DR signalling.
| Identifer | oai:union.ndltd.org:ADTP/254180 |
| Creators | Keyur Dave |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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