Nedd8 Regulation of Ubiquitin E3 Ligases in Adipocytes

描述(申请人提供)：病态肥胖症和儿童肥胖症的特征是脂肪组织块过度积累，这是由于现有分化的脂肪细胞扩大(肥厚性肥胖)，以及由于前脂肪细胞的增殖和随后的分化而获得新的脂肪细胞(增生性肥胖)。考虑到儿童肥胖率的快速上升和对这些人的破坏性长期健康影响，更多地了解调控脂肪细胞增殖的分子机制变得至关重要。几年来，我们的实验室一直专注于细胞周期蛋白依赖性激酶抑制物的调节和功能，如p27和p21，它们控制着细胞周期从G1期到S期的进程，作为前体脂肪细胞复制的分子调节因子。这项研究的初步数据表明，SCFSkp2 E3连接酶在调节脂肪细胞增殖过程中26S蛋白酶体对p27的多泛素化和降解过程中具有生理作用。此外，我们的初步数据表明，在脂肪细胞中，SCF E3连接酶的蛋白质亚单位Cul1被泛素样分子Nedd8修饰，并且在生理条件下，Nedd8-Cul1结合的程度在G1期进程中受到调节。基于这些数据和已发表的文献，我们提出了一个中心假设，即Nedd8修饰SCF E3连接酶是一个动态过程，涉及将Nedd8与其cullin蛋白靶标连接和移除的相反途径，并且这两条途径对于泛素介导的p27蛋白降解和脂肪细胞增殖是必不可少的。我们将在两个目标上检验中心假说。第一部分探讨了G1期Nedd8修饰对E3连接酶活性的调节的生理意义和机制。第二个决定了CSN作为Nedd8异肽酶的作用，能够去除Nedd8，以及对细胞SCF活性的影响。这两个目标都是通过评估可测试的工作模式来解决连接NED8和移除NED8的机制。我们预测，负责添加和移除Nedd8的途径是SCF E3连接酶活性、p27泛素化和降解以及脂肪细胞增殖所必需的。

DESCRIPTION (provided by applicant): Morbid and childhood obesity are characterized by excessive accumulation of adipose-tissue mass that results from enlargement of existing differentiated adipocytes (hypertrophic obesity) and from acquisition of new adipocytes from proliferation and subsequent differentiation of preadipocytes (hyperplastic obesity). Considering the rapid rise in childhood obesity and the devastating long term heath implications to these individuals, it has become critically important to gain greater knowledge of the molecular mechanisms regulating adipocyte hyperplasia. For several years, our lab has focused on the regulation and function of cyclin-dependent kinase (CDK) inhibitors, such as p27 and p21 that control G1 to S phase progression of the cell cycle as molecular regulators of preadipocyte replication. Preliminary data of this proposal demonstrate a physiological roll for the SCFSkp2 E3 ligase in mediating polyubiquitylation and degradation of p27 by the 26S proteasome during adipocyte hyperplasia. Furthermore, our preliminary data demonstrate that Cul1, a protein subunit of the SCF E3 ligase, is modified by the ubiquitin-like molecule Nedd8 in adipocytes and that the extent of Nedd8-Cul1 conjugates is regulated during G1 phase progression under physiological conditions. Based on these data and published literature, we developed the central hypothesis that Nedd8 modification of the SCF E3 ligase is a dynamic process that involves opposing pathways that attach and remove Nedd8 from its cullin protein target and that both pathways are essential for ubiquitin-mediated p27 protein degradation and adipocyte hyperplasia. We will test the central hypothesis in two aims. The first explores the physiological significance and mechanism of regulated Nedd8 modification during G1 phase progression regarding E3 ligase activity. The second determines the role of the CSN as a Nedd8 isopeptidase capable of removing Nedd8 and the impact regarding cellular SCF activity. Both aims address mechanisms linking attachment and removal of Nedd8 through evaluation of testable working models. We predict that pathways responsible for adding and removing Nedd8 are essential for SCF E3 ligase activity, p27 ubiquitylation and degradation, and adipocyte hyperplasia.