Drosophila nuclear receptor proteomics

Nuclear receptors comprise a large family of important and versatile DNA binding transcription factors. At their carboxy-termini are modular domains that have the ability to function as molecular switches that change the activity and specificity of the associated receptor. Those with known ligands bind relatively small (200-600 daltons) hydrohphobic molecules, many of which are steroids that diffuse readily within and between tissues. The ability to respond to such chemicals empowers these receptors with the capacity to coordinate tissue patterning, differentiation and growth in response to remotely synthesized molecules. The importance of nuclear receptors in normal development, metabolism and oncogenesis also makes them important potential targets for drug treatment, manipulation and intervention.

Our work, done in collaboration with Henry Krause and Cheryl Arrowsmith and funded by GlaxoSmithKline and NCIC, focuses specifically on Drosophila nuclear receptors and the use of developing Drosophila tissues as a comprehensive test bed for nuclear receptor, cofactor and ligand function. We have identified 16 classic nuclear receptors in the sequenced Drosophila genome. All 16 receptors are studied in parallel in a series of complementary in vitro and in vivo approaches.

The principal aims of our work are:
• Subcloning all 16 receptors into the expression and reporter constructs required for this study.
• Expression purification of the ligand binding domains of each NR in bacteria for X-ray crystallography and NMR structure analysis.
• Expression of the full-length proteins in flies with N-terminal TAP tags in order to facilitate the isolation and identification of associated cofactors and ligands.
• Development and use of a “ligand trap” reporter system as a functional readout for the localization and testing of potential ligands and cofactors in developing flies and cultured cells.
• Exploration of the use of flies as a screening bed for human nuclear receptor ligands and cofactors.

Of the 16 classic nuclear receptors that we have identified, only one has a known ligand and only three have known cofactors. Our work has the potential to identify physiological and synthetic ligands. We are also trying to identify and assess potential cofactors. Once ligands are identified, it will be possible to test or design new reagents that mimic their structures and that may serve as agonists, antagonists or tissue-specific modifiers. Similarly, the identification of cofactors and their modes of interaction will provide new and novel ways to manipulate these proteins. Hence, these studies should lead rather directly and quickly to the design of therapeutic reagents for disease treatment and prevention.

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