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: 7676844
• 负责人: Michael P Rape
• 金额: $ 29.17万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676844
• 关键词: 26S proteasome; Accounting; Address; Affect; Binding; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Extracts; Cells; Collaborations; Competence; Complex; Conserved Sequence; DNA Damage; Deubiquitinating Enzyme; Deubiquitination; Development; Enzymes; Eukaryota; Event; Genetic Screening; Goals; Human; In Vitro; Lead; Life; Light; Malignant Neoplasms; Mitosis; Mitotic; Modification; Mutate; Paclitaxel; Phosphoric Monoester Hydrolases; Phosphotransferases; Post-Translational Protein Processing; Proteins; Proto-Oncogenes; Reaction; Regulation; Reporting; Resistance; Role; Somatic Cell; Substrate Specificity; System; Testing; Therapeutic Intervention; Time; Tissues; Ubiquitin; Ubiquitination; anaphase-promoting complex; cancer cell; cell growth regulation; in vitro Assay; in vivo; inhibitor/antagonist; insight; novel; protein degradation; reconstitution; research study; response; small molecule; tumorigenesis; ubiquitin ligase

DESCRIPTION (provided by applicant): The specific degradation of proteins by the 26S proteasome is a key mechanism in cell cycle control. Proteins to be degraded are modified with a ubiquitin chain, which is catalyzed by ubiquitin ligases. By contrast, specific deubiquitinating enzymes can remove ubiquitin chains and thereby rescue proteins from degradation. The misregulation of ubiquitination and deubiquitination can cause aberrant proliferation and cancer. It is our long term goal to understand how the dynamic modification of proteins with ubiquitin controls cell cycle progression, and how it is misregulated in cancer. To address these questions, we focus on the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C). The APC/C is essential for proliferation in all eukaryotes. It orchestrates progression through mitosis by promoting the degradation of cell cycle regulators in a highly conserved sequence, which is referred to as substrate ordering. The APC/C achieves substrate ordering by catalyzing the formation of ubiquitin chains with different degrees of processivity. The more processive the ubiquitination of an APC/C-substrate, the earlier it is degraded in mitosis. The activity of the APC/C and the timing of degradation of APC/C-substrates during mitosis are regulated by specific deubiquitinating enzymes. Therefore, a tight interplay between ubiquitin chain formation by the APC/C and deubiquitination by specific deubiquitinating enzymes accounts for the controlled progression of cells through mitosis. In specific aim (1) of this application, we will dissect the mechanisms that underlie ubiquitin chain formation by the APC/C, and thus, substrate ordering. In specific aim (2), we will reconstitute deubiquitinating enzymes that regulate APC/C-activity and substrate degradation. Dissecting the substrate specificity and regulation of these enzymes will allow us to determine the importance of deubiquitination for cell cycle regulation and could lead to the identification of new cell cycle regulators. Finally, in specific aim (3), we will test the hypothesis that the co-regulation of ubiquitin ligases and deubiquitinating enzymes can account for multiple layers of cell cycle regulation, including the transient inactivation of cell cycle regulators, in the absence of proteasomal degradation. We will use a complementary approach of mechanistic studies in vitro and live-cell analysis in vivo to address these important questions. Our experiments will shed light onto fundamental aspects of cell cycle control and could identify new cell cycle regulators. Importantly, the misregulation of the APC/C during early mitosis can underlie the resistance of cancer cells against treatment with the chemotherapeutic taxol. Our reconstitution of key cell cycle reactions in vitro could lay the groundwork for screens aimed at identifying small molecules that specifically inhibit APC/C and restore the responsiveness of cancer cells to taxol treatment.

描述（由申请方提供）：26 S蛋白酶体对蛋白质的特异性降解是细胞周期控制的关键机制。待降解的蛋白质被泛蛋白链修饰，该链由泛蛋白连接酶催化。相比之下，特异性去泛素化酶可以去除泛素链，从而拯救蛋白质免于降解。泛素化和去泛素化的失调可导致异常增殖和癌症。我们的长期目标是了解泛素对蛋白质的动态修饰如何控制细胞周期进程，以及它如何在癌症中被错误调节。为了解决这些问题，我们专注于泛素连接酶后期促进复合物/环体（APC/C）。APC/C是所有真核生物增殖所必需的。它通过促进高度保守序列中细胞周期调节因子的降解来协调有丝分裂的进程，这被称为底物排序。APC/C通过催化形成具有不同程度的持续合成能力的泛素链来实现底物有序化。APC/C-底物的泛素化越进行，其在有丝分裂中降解越早。APC/C的活性和APC/C底物在有丝分裂期间的降解时间由特异性去泛素化酶调节。因此，由APC/C形成的泛素链和由特异性去泛素化酶进行的去泛素化之间的紧密相互作用解释了细胞通过有丝分裂的受控进展。在本申请的具体目标（1）中，我们将剖析APC/C形成泛素链的机制，从而剖析底物排序。在具体目标（2）中，我们将重组调节APC/C活性和底物降解的去泛素化酶。剖析这些酶的底物特异性和调节将使我们能够确定去泛素化对细胞周期调节的重要性，并可能导致新的细胞周期调节因子的鉴定。最后，在具体的目标（3）中，我们将检验这样的假设，即在没有蛋白酶体降解的情况下，泛素连接酶和去泛素化酶的共同调节可以解释细胞周期调控的多层，包括细胞周期调控因子的瞬时失活。我们将使用体外机制研究和体内活细胞分析的互补方法来解决这些重要问题。我们的实验将揭示细胞周期控制的基本方面，并可以确定新的细胞周期调节剂。重要的是，在早期有丝分裂过程中APC/C的错误调节可能是癌细胞对化疗紫杉醇治疗的抗性的基础。我们对关键细胞周期反应的体外重建可以为旨在鉴定特异性抑制APC/C的小分子并恢复癌细胞对紫杉醇治疗的反应性的筛选奠定基础。