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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 A.具体目标 2008年3月，外部评审员指出，“弗洛伊德博士的主要问题是关注长期最具生产力和可持续性的领域”，“重要的是要确定靶向泛素蛋白酶体系统的PPARg具有调节意义”。 实现这些目标的建议包括将更多的重点放在识别和表征泛素连接酶的目标PPARg降解使用池siRNA库，而不是我们原来的计划，使用基于质谱的方法。 除了考虑大规模的无偏连接酶筛选外，评审员建议我们开发一个可能的候选人的短列表，以评估作为靶向PPARg降解的潜在连接酶。 评审员还指出，“确定PPARg在脂肪组织中的半衰期，然后清楚地证明由于降解速率的改变而导致的蛋白质浓度变化将是确定泛素蛋白酶体系统是否确实是一个重要的调节点的良好开端”。 具体目标1：检验脂肪细胞中的PPARg被特异性E3连接酶直接靶向进行泛素依赖性降解的假设。 我们将使用基于脂肪细胞的siRNA筛选来鉴定负责调节PPARg稳定性和PPARg遍在化的遍在蛋白E3连接酶（或多个连接酶）。 该筛选将使我们能够确定靶向PPARg降解的E3连接酶/s是否是脂肪细胞中核受体辅助调节因子的组分。 具体目标二：检验配体诱导的PPARg泛素化与PPARg转录激活和随后的PPARg蛋白酶体依赖性降解相关的假设。 我们将使用表达天然存在的PPARg错义突变的稳定细胞系沿着PPARg泛素化、稳定性和SUMO化的生化测定。 这一目标也将测试PPARg的SUMO化影响PPARg泛素化的假设。 具体目标3：检验营养和代谢信号调节PPARg的泛素和SUMO-1修饰以及PPARg稳定性的假设。 为此，我们将使用饮食诱导的肥胖小鼠模型与基于细胞培养的脂肪细胞模型相结合。

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.