NEDD8: Mechanisms of conjugation and identification of binding partners

小鼠遗传研究表明，环结构域蛋白Mdm2是肿瘤抑制因子p53的主要拮抗剂。在生化水平上，Mdm2同时作为p53的E3泛素蛋白连接酶和E3 nedd8蛋白连接酶，从而调节p53的亚细胞定位、稳定性和转录反激活特性。然而，尽管许多研究小组对p53的泛素化进行了广泛的研究，但对Mdm2介导的p53类化作用的机制以及类化作用影响p53功能的机制知之甚少。在目前的资助期内，我们已经确定了mdm2相关蛋白MdmX作为mdm2介导的类化修饰的底物，并表明以前未表征的蛋白NCE2是nedd8偶联酶。在本应用中，我们建议继续努力鉴定参与Mdm2介导的p53和MdmX类化修饰的蛋白，并深入了解Mdm2是否诱导泛素化或类化修饰相关蛋白的机制。此外，nedd8偶联酶NCE2和UBC12将在功能上进一步表征，基于亲和的纯化方法将用于鉴定选择性结合并最终决定类化蛋白命运的蛋白质。综上所述，拟议的研究将有助于阐明mdm2介导的类化修饰特别是蛋白质类化修饰的机制。

Genetic studies in mice have shown that the RING domain protein Mdm2 is a major antagonist of the tumor suppressor p53. At the biochemical level, Mdm2 functions as both an E3 ubiquitin-protein ligase and E3 NEDD8-protein ligase for p53 thereby regulating subcellular localization, stability, and the transcriptional transactivation properties of p53. However, while ubiquitylation of p53 has been extensively studied by many groups, only little is known about the mechanisms involved in Mdm2- mediated neddylation of p53 as well as the mechanism, by which neddylation affects p53 function. In the current funding period, we have identified the Mdm2-related protein MdmX as a substrate for Mdm2-mediated neddylation and showed that the previously uncharacterized protein NCE2 is a NEDD8-conjugating enzyme. In this application, we propose to continue our efforts to identify proteins that are involved in Mdm2-mediated neddylation of p53 and MdmX and to obtain insight into the mechanisms that determine whether Mdm2 induces ubiquitylation or neddylation of associated proteins. In addition, the NEDD8-conjugating enzymes NCE2 and UBC12 will be functionally further characterized and affinity-based purification approaches will be employed to identify proteins that selectively bind to and eventually determine the fate of neddylated proteins. Taken together, the proposed studies will contribute to the elucidation of the mechanisms involved in Mdm2-mediated neddylation in particular and in neddylation of proteins in general.