Dissecting Mechanisms of Ubiquitination and Deubiquitination in Cell Cycle Contro

描述（由申请人提供）：泛素链的翻译后修饰对于所有真核生物的细胞分裂是必不可少的。泛素链由E1泛素激活酶、E2泛素缀合酶和E3泛素连接酶级联反应合成，后者定义了这种修饰的底物特异性。在约600种人类E3中，后期促进复合物（APC/C）对于有丝分裂是必需的，并且APC/C调节剂或底物的异常表达与肿瘤发生紧密相关。此外，紫杉醇（一种激活APC/C抑制性纺锤体检查点的小分子）对APC/C的间接抑制已成为化疗的支柱。我们最近发现，APC/C装配链的一种新的拓扑结构，通过Lys 11的泛素连接。APC/C通过使用起始E2（Ube 2C）和延长E2（Ube 2S）来实现K11-连接形成。与APC/C类似，K11连接的链和由Ube 2C和Ube 2S组成的E2模块是细胞分裂所必需的。因此，解剖APC/C依赖的组装和K11连接链的功能对我们理解细胞分裂至关重要。在这里，我们建议通过使用一套独特的生化和结构分析，结合广泛的生化筛选得到的泛素和E2突变体，来剖析K11连接的泛素链形成的机制。我们将通过采用E3重编程的新方法直接测试K11连接的泛素链的功能，该方法允许我们切换由APC/C在细胞中组装的泛素链的拓扑结构。最后，我们将研究K11连接链形成的调节，使用一组癌症相关的APC/C-底物，纺锤体组装因子HURP，NuSAP和Tpx 2，作为我们的模型系统。总之，我们希望我们的研究将揭示泛素化和细胞周期调控的核心原则。以这种方式，我们的工作将可能为开发靶向必需E2酶的新型化疗药物提供指导。

DESCRIPTION (provided by applicant): Posttranslational modification with ubiquitin chains is essential for cell division in all eukaryotes. Ubiquitin chains are synthesized by a cascade of E1 ubiquitin-activating, E2 ubiquitin-conjugating, and E3 ubiquitin ligase enzymes, with the latter defining the substrate specificity of this modification. Among ~600 human E3s, the anaphase-promoting complex (APC/C) is essential for mitosis, and the aberrant expression of APC/C- regulators or substrates has been tightly linked to tumorigenesis. Moreover, the indirect inhibition of the APC/C by paclitaxel, a small molecule that activates the APC/C-inhibitory spindle checkpoint, has become a mainstay of chemotherapy. We have recently discovered that the APC/C assembles chains of a novel topology, linked through Lys11 of ubiquitin. The APC/C achieves K11-linkage formation by employing an initiating E2, Ube2C, and an elongating E2, Ube2S. Similar to the APC/C, K11-linked chains and the E2-module composed of Ube2C and Ube2S are required for cell division. Therefore, dissecting the APC/C-dependent assembly and function of K11-linked chains is critical to our understanding of cell division. Here, we propose t dissect the mechanism of K11-linked ubiquitin chain formation by using a set of unique biochemical and structural assays combined with ubiquitin- and E2-mutants derived from extensive biochemical screens. We will directly test for functions of K11-linked ubiquitin chains by employing the novel approach of E3-reprogramming that allows us to switch the topology of ubiquitin chains that are assembled by the APC/C in cells. Finally, we will investigate the regulation of K11-linked chain formation, using a group of cancer-related APC/C-substrates, the spindle assembly factors HURP, NuSAP, and Tpx2, as our model system. Together, we expect that our studies will reveal core principles of ubiquitylation and cell cycle regulation. In this manner, our work will likely provide guidance for the development of novel classes of chemotherapeutics that target essential E2 enzymes.