Dissecting Mechanisms of Ubiquitination and Deubiquitination in Cell Cycle Contro

描述(申请人提供)：在所有真核生物中，泛素链的翻译后修饰对于细胞分裂是必不可少的。泛素链是由E1泛素激活酶、E2泛素结合酶和E3泛素连接酶串联而成的，后者决定了这种修饰的底物特异性。在人类约600个E3中，后期促进复合体(APC/C)是有丝分裂所必需的，APC/C调节因子或底物的异常表达与肿瘤的发生密切相关。此外，紫杉醇对APC/C的间接抑制已成为化疗的主要手段。紫杉醇是激活APC/C抑制纺锤体检查点的小分子。我们最近发现，APC/C组装了一种新的拓扑链，通过泛素的Lys11连接在一起。APC/C通过使用起始的E2，Ube2C和延伸的E2，UBE2S来实现K11-连锁形成。与APC/C类似，细胞分裂也需要K11连接链和由Ube2C和UBE2S组成的E2模块。因此，解剖依赖于APC/C的K11连接链的组装和功能对于我们理解细胞分裂是至关重要的。在这里，我们建议通过一系列独特的生化和结构分析结合来自广泛的生化筛选的泛素和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-重编程的新方法来直接测试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。最后，我们将使用一组与癌症相关的APC/C底物、纺锤体组装因子HURP、NuSAP和Tpx2作为我们的模型系统来研究K11连接链的形成规律。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理。通过这种方式，我们的工作可能会为开发以必需的E2酶为靶点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.