Dissecting Mechanisms of Ubiquitination and Deubiquitination in Cell Cycle Contro

剖析细胞周期控制中泛素化和去泛素化的机制

• 批准号: 8593301
• 负责人: Michael P Rape
• 金额: $ 25.69万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8593301
• 关键词: Address; Aneuploidy; Biochemical; Biological; Biological Assay; Biological Models; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell division; Cells; Code; Congenital Abnormality; Coupled; DNA Damage; Deubiquitination; Development; Disease; Drug Targeting; Enzymes; Eukaryota; Foundations; Funding; Future; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Lead; Left; Link; Malignant Neoplasms; Microtubules; Mitosis; Mitotic; Modification; Molecular; Mutate; Paclitaxel; Phosphotransferases; Post-Translational Protein Processing; Process; Property; Regulation; Role; Specificity; Substrate Specificity; Testing; Time; Ubiquitin; Ubiquitination; Work; Yeasts; anaphase-promoting complex; base; chemotherapy; human disease; insight; mutant; novel; novel strategies; public health relevance; research study; small molecule; tumorigenesis; ubiquitin-protein ligase

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.

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至关重要。泛素链由 E1 泛素激活酶、E2 泛素结合酶和 E3 泛素连接酶级联合成，后者定义了该修饰的底物特异性。在约 600 个人类 E3 中，后期促进复合物 (APC/C) 对于有丝分裂至关重要，并且 APC/C 调节剂或底物的异常表达与肿瘤发生密切相关。此外，紫杉醇（一种激活 APC/C 抑制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 组装了一种新颖的拓扑链，通过泛素的 Lys11 连接。 APC/C 通过使用起始 E2、Ube2C 和延伸 E2、Ube2S 实现 K11 连接形成。与 APC/C 类似，细胞分裂需要 K11 连接链和由 Ube2C 和 Ube2S 组成的 E2 模块。因此，剖析 K11 连接链的 APC/C 依赖性组装和功能对于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测定结合广泛生化筛选中衍生的泛素和 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细胞中 APC/C 组装的泛素链的拓扑结构。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，研究 K11 连接链形成的调节。我们共同期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针对必需 E2 酶的新型化疗药物提供指导。