Nedd8 Regulation of Ubiquitin E3 Ligases in Adipocytes

描述（由申请人提供）：病态和儿童期肥胖的特点是脂肪组织团块的过度积累，这是由于现有已分化的脂肪细胞扩大（肥厚性肥胖），以及由于前脂肪细胞增殖和随后的分化而获得新的脂肪细胞（增生性肥胖）。考虑到儿童肥胖的快速增长和对这些个体的破坏性长期健康影响，获得更多的脂肪细胞增生调节分子机制的知识变得至关重要。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（CDK）抑制剂的调控和功能，如p27和p21，它们控制细胞周期的G1到S期进展，作为前脂肪细胞复制的分子调节剂。本提案的初步数据证明了SCFSkp2 E3连接酶在脂肪细胞增生过程中介导p27的多泛素化和26S蛋白酶体降解的生理作用。此外，我们的初步数据表明，在脂肪细胞中，SCF E3连接酶的一个蛋白质亚基Cul1被泛素样分子Nedd8修饰，并且在生理条件下，Nedd8-Cul1偶联的程度在G1期进展过程中受到调节。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SCF E3连接酶的Nedd8修饰是一个动态过程，涉及到从cullin蛋白靶标上附着和去除Nedd8的相反途径，这两种途径对于泛素介导的p27蛋白降解和脂肪细胞增生都是必不可少的。我们将从两个方面检验中心假设。第一部分探讨了G1期调控的Nedd8修饰对E3连接酶活性的生理意义和机制。第二个决定了CSN作为能够去除Nedd8的Nedd8异肽酶的作用以及对细胞SCF活性的影响。这两个目标都是通过评估可测试的工作模型来解决连接Nedd8附着和移除的机制。我们预测，负责添加和去除Nedd8的途径对于SCF E3连接酶活性、p27泛素化和降解以及脂肪细胞增生至关重要。

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.