The nuclear receptor superfamily is responsible for regulating the expression of genes involved in development, reproduction and metabolism. These transcription factors control the expression of their target genes through the binding of small molecule regulators to their ligand binding domains. Classical nuclear receptors include the steroid receptors, which bind endocrine hormones and have been important targets of pharmaceutical intervention. However, approximately one half of the human nuclear receptors remain orphans and are without known cognate ligands.
Focusing on the Drosophila orthologues of these orphan receptors, this project used mass spectrometry to identify the chemical diversity associated with the receptors following expression in recombinant systems. In a genome-wide screen of Drosophila nuclear receptors, this approach identified co-purifying molecules with a number of receptors. The physiological relevance of these putative ligand/receptor pairs was determined through biochemical analysis, in vivo characterization and structure determination. Ligand(s) or the ligand state was identified for the Drosophila receptors: DHR3, DHR96, E75, Ftz-f1 and USP. Of these, three were validated through the efforts of this project, and independent groups confirmed the remaining two. The most significant findings were the discoveries that the fly nuclear receptor E75 is regulated by heme, gas and redox, and that there is a similar regulatory scheme in the human orthologues, Reverbα and β. Furthermore, crystallization of the heme-bound Rev-erbβ ligand binding domain was also achieved, and this provided key insights into the mechanism of ligand regulation for the Rev-erbs.
This project highlighted the role of nuclear receptors in metabolic surveillance. The ligands/signals identified in association with these receptors include: cholesterol, dehydrocholesterol, heme, NO, CO, redox and phospholipids. Unlike the classical steroid hormones, these are not dedicated signaling molecules, but instead are key substrates or products of metabolism. In the context of nuclear receptor signaling, I hypothesize that these metabolites serve as metabolic indicators/signals in the regulation of development and metabolism. Furthermore, four of these Drosophila receptors comprise the ecdysone-response pathway in the developing fly. Taken together, this suggests that both the metabolic state of the organism and steroid hormones drive nuclear receptor regulation of development.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/24844 |
| Date | 01 September 2010 |
| Creators | Pardee, Keith Ian |
| Contributors | Edwards, Aled M. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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