P3: REGULATION OF PPARGAMMA IN ADIPOCYTES BY THE UBIQUITIN PROTEASOME SYSTEM

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims In March 2008, the external reviewers noted that the "major issue for Dr Floyd is focusing on the area that will be most productive and sustainable over the long-term" and that "it is important to establish that the PPARg targeted to the ubiquitin proteasome system has regulatory significance". Recommendations for achieving these goals included placing more focus on identifying and characterizing ubiquitin ligases that target PPARg for degradation using a pooled siRNA library rather than our original plan to use a mass spectroscopic-based approach. In addition to considering a large-scale unbiased ligase screen, the reviewers suggested that we develop a short list of possible candidates to evaluate as potential ligases targeting PPARg for degradation. The reviewers also noted that "determining the half-life of PPARg in adipose tissue and then demonstrating clearly that the concentration of the protein changes due to altered rates of degradation would be a good start in establishing whether the ubiquitin proteasome system is indeed an important regulatory point". Specific Aim 1: Test the hypothesis that PPARg in adipocytes is directly targeted by specific E3 ligases for ubiquitin-dependent degradation. We will identify the ubiquitin E3 ligase (or ligases) responsible for regulating PPARg stability and PPARg ubiquitylation using adipocyte-based siRNA screening. This screen will allow us to determine if the E3 ligase/s targeting PPARg for degradation are components of the nuclear receptor coregulators in adipocytes. Specific Aim 2: Test the hypothesis that ligand-induced ubiquitylation of PPARg is associated with both PPARg transcriptional activation and subsequent proteasome-dependent degradation of PPARg. We will use stable cell lines expressing naturally occurring missense mutations of PPARg along with biochemical assays of PPARg ubiquitylation, stability, and SUMOylation. This aim will also test the hypothesis that SUMOylation of PPARg influences PPARg ubiquitylation. Specific Aim 3: Test the hypothesis that nutrition and metabolic signals regulate ubiquitin and SUMO-1 modification of PPARg as well as PPARg stability. We will use murine models of diet-induced obesity coupled with cell culture-based models of adipocytes in this aim.