Peroxisome Proliferator-Activated Receptors (PPARs) belong to the nuclear receptor superfamily of transcription factors. PPARs, like some other nuclear receptors, bind their cognate DNA binding sites as heterodimers with Retinoid X Receptor (RXR). Like most members of this superfamily, PPARs regulate transcription of target genes in response to specific lypophilic ligands. Given the fact, however, that many members of the nuclear receptor superfamily possess a ligand independent activation function (AF -1) in their amino-terminal A/B domains, we identified and characterized the AF-1 activity in the A/B domains of mPPARα and mPPARγ2. When fused to the GAL4 DNA Binding
Domain (DBD), the A/B domains of both mPPARα and mPPARγ2 subtypes are transcriptionally active both in mammalian and yeast cells in the absence of cognate PPAR activators, indicating that both PPAR subtypes do in fact contain AF -1 activities in their respective A/B domains. Our deletion studies localizing the region in the A/B domains of the PPAR subtypes indicate that the amino-terminal region of A/B domains of both PP AR subtypes is necessary, but not sufficient, for the AF -1 activity. After having identified and characterized the AF-1 activities of both mPPARα and mPPARγ subtypes, we proceeded to investigate how this activity is controlled. Generally, nuclear receptors, including PPARs, do not activate transcription when ligand is absent, suggesting that the ligand-independent AF -1 activity is somehow masked. We used the GAL4 DBD to investigate this phenomenon. When fused to GAL4 DBD, the full-length mPPARα receptor is not active in mammalian cells in absence of ligand despite the presence of the AF -1 activity in the A/B domain. Only when the PPAR activator Wy-14 is added, does the receptor become active. Carboxy-terminal deletion
of the fusion at the junction of the DBD (C domain) and the Hinge region (D domain)
results in partially active protein whose activity does not depend on the presence of ligand, suggesting a role of the hinge region in the masking of the AF-1 activity. The transcriptional activity of PPARs is ligand independent when PPARs are expressed as GAL4 DBD fusions or allowed to bind reporter genes as dimers with RXR. Yeast do not possess nuclear receptors or any homologues of known accessory proteins which interact with nuclear receptors. Given our observations and those of others made in yeast and mammalian cells, we suggest that the masking of the AF -1 activity occurs in the inactive state of the receptor, and that mammalian cells contain unidentified factor(s)
which is able to maintain PPARs in the inactive state, thus enabling the masking of the AF-1 activity.
Nuclear receptors including PPARs are known to interact with a host of accessory proteins which modulate their activity and are thought to act as a bridge between the receptors and the basal transcription machinery. One of these proteins is RIP140 whose function has not been determined. Our studies suggest that RIP 140 is a co-activator for the PPAR/RXR heterodimer, as well as for the AF-1 activity of mPPARα and mPP ARγ2. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22682 |
| Date | 05 1900 |
| Creators | Niemkiewicz, Michal |
| Contributors | Capone, John, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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