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

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 2009年3月，外部评价者指出，我们的项目“有可能揭示新的机制洞察力”来调控PPARγ，我们对泛素E3连接酶的初步筛选发现，“E3连接酶似乎影响细胞内PPARγ的稳定水平”。评审员还建议，“在这一领域成功竞争R01可能需要将重点缩小到一种或两种在PPAR-Gamma作用中发挥作用的酶”。一个主要的担忧仍然是将PPARγ泛素化和降解与PPAR伽马转录功能或下游代谢改变联系起来。实现这些目标的建议集中在继续我们的E3连接酶实验，专注于一种或两种连接酶，确定E3连接酶对PPAR伽马蛋白水平的影响是直接的还是间接的，并考虑选定的E3连接酶对脂肪形成的影响。此外，评价者建议我们重点关注PPARGamma的泛素蛋白酶体修饰，作为PPARGamma的一种受调控的翻译后修饰。 因应评审员的建议，我们达致以下目标： 具体目标1：完成二次筛选以确定调节成熟脂肪细胞中PPARγ蛋白水平的泛素E3连接酶。 特定目的2：检测所选择的一个或多个连接酶对配体增强的泛素化和蛋白酶体依赖的PPARγ降解的影响。 具体目标3：确定已鉴定的一个或多个连接酶对脂肪形成的影响。

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 2009, the external reviewers indicated that our project "has the potential to reveal new mechanistic insights" into the regulation of PPARgamma and that our initial screen of ubiquitin E3 ligases has identified "E3 ligases that appear to influence steady state levels of PPARgamma within cells". The reviewers also advised that "successfully competing for an R01 in this area is likely to require narrowing the focus to one or two enzymes that play functional roles in PPARgamma action". A primary concern continued to be linking PPARgamma ubiquitylation and degradation to PPARgamma transcriptional function or a down-stream metabolic alteration. Recommendations for achieving these goals centered on continuing our experiments with the E3 ligases by focusing on one or two ligases, determining whether the effect of the E3 ligase on PPARgamma protein levels is direct or indirect and considering the effect of the selected E3 ligase on adipogenesis. In addition, the reviewers recommended we focus on ubiquitin proteasome modification of PPARgamma as a regulated posttranslational modification of PPARgamma. In response to the reviewers' suggestions, we pursued the following aims: Specific Aim 1: Complete the secondary screen to identify ubiquitin E3 ligases that regulate PPARgamma protein levels in mature adipocytes. Specific Aim 2: Examine the effect of the selected ligase or ligases on ligand-enhanced ubiquitylation and proteasome-dependent degradation of PPARgamma. Specific Aim 3: Determine the effect of the identified ligase or ligases on adipogenesis.