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

P3：泛素蛋白酶体系统对脂肪细胞中 PPARGAMMA 的调节

• 批准号: 7959986
• 负责人: ZELPHA ELIZABETH FLOYD
• 金额: $ 22.65万
• 依托单位: LSU PENNINGTON BIOMEDICAL RESEARCH CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959986
• 关键词: Adipocytes; Adipose tissue; Area; Biochemical; Biological Assay; Cell Culture Techniques; Computer Retrieval of Information on Scientific Projects Database; Coupled; Diabetes Mellitus; Diet; Funding; Goals; Grant; Half-Life; Institution; Libraries; Ligands; Ligase; Louisiana; Mentors; Metabolic; Missense Mutation; Modeling; Modification; Mus; Nuclear Receptors; Obesity; PPAR gamma; Proteins; Recommendation; Regulation; Research; Research Personnel; Resources; Screening procedure; Signal Transduction; Small Interfering RNA; Source; System; Testing; Transcriptional Activation; Ubiquitin; United States National Institutes of Health; base; multicatalytic endopeptidase complex; nutrition; stable cell line; ubiquitin ligase; ubiquitin-protein ligase

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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 A.具体目标 2008年3月，外部审查员指出，“弗洛伊德博士面临的主要问题是关注长期最具生产力和可持续性的领域”，“重要的是要确定针对泛素蛋白酶体系统的PPARg具有调控意义”。实现这些目标的建议包括更多地关注使用汇集的siRNA文库来识别和表征针对PPARg降解的泛素连接酶，而不是我们最初计划使用基于质谱学的方法。除了考虑大规模的无偏连接酶筛选外，评价者建议我们开发一份可能的候选短名单，作为针对PPARg降解的潜在连接酶的评估。 审查人员还指出，“确定PPARg在脂肪组织中的半衰期，然后清楚地证明蛋白质的浓度因降解速度的变化而变化，将是确定泛素蛋白酶体系统是否确实是一个重要的调控点的良好开端”。 具体目标1：验证脂肪细胞中的PPARg直接被特定的E3连接酶靶向进行泛素依赖的降解的假设。我们将通过基于脂肪细胞的siRNA筛选来鉴定负责调节PPARg稳定性和PPARg泛素化的泛素E3连接酶。这一筛选将使我们能够确定针对PPARg降解的E3连接酶/S是否是脂肪细胞中核受体协同调节因子的组成部分。 特定目的2：验证配体诱导的PPARg泛素化与PPARg转录激活和随后的蛋白酶体依赖的PPARg降解相关的假设。我们将使用稳定的细胞株表达自然发生的PPARg错义突变，以及PPARg泛素化、稳定性和SUMO化的生化分析。这一目的也将检验PPARg的SUMO化影响PPARg泛素化的假设。 具体目标3：验证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。在这一目标中，我们将使用饮食诱导肥胖的小鼠模型和基于细胞培养的脂肪细胞模型。