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: 8102982
• 负责人: Michael P Rape
• 金额: $ 26.98万
• 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 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8102982
• 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（由申请人提供）：26S蛋白酶体对蛋白质的特定降解是细胞周期控制中的关键机制。要降解的蛋白质用泛素链修饰，泛素链被泛素连接酶催化。相比之下，特定的去泛素化酶可以去除泛素链，从而从降解中挽救蛋白质。泛素化和去泛素化的正调会导致异常的增殖和癌症。我们的长期目标是了解用泛素的蛋白质动态修饰如何控制细胞周期的进展以及如何在癌症中误导。为了解决这些问题，我们专注于泛素连接酶后期的复合体/循环体（APC/C）。 APC/C对于所有真核生物的增殖至关重要。它通过在高度保守的序列中促进细胞周期调节剂的降解来策划通过有丝分裂的进展，该序列称为底物排序。 APC/C通过催化不同程度的加工性来催化泛素链的形成来实现底物排序。 APC/C-基底物的泛素化越多，其在有丝分裂中的降解越早。 APC/C的活性以及在有丝分裂过程中APC/C-底物降解的时间通过特定的去泛素化酶来调节。因此，APC/C的泛素链形成与特定去泛素化酶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控制进展。在本应用的特定目的（1）中，我们将剖析APC/C的泛素链形成基础的机制，从而剖析底物排序。在特定目的（2）中，我们将重新建立调节APC/C活性和底物降解的酶。解剖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，并可能导致鉴定新的细胞周期调节剂。最后，在特定的目的（3）中，我们将检验以下假设：泛素连接酶和去泛素化酶的共调节可以解释蛋白酶体去降解的多个细胞周期调节，包括细胞周期调节剂的瞬时灭活。我们将在体外使用机械研究的补充方法，并在体内使用活细胞分析来解决这些重要问题。我们的实验将启用细胞周期控制的基本方面，并可以鉴定新的细胞周期调节剂。重要的是，在早期有丝分裂期间，APC/C的正调可能是癌细胞抵抗化学治疗紫杉醇治疗的抗性。我们在体外重新建立了关键细胞周期反应，可以为旨在识别专门抑制APC/C的小分子的筛选奠定基础，并恢复癌细胞对紫杉醇治疗的反应性。