Molecular and functional analysis of the APC/C ubiquitin ligase complex using the MultiBac system

泛素介导的蛋白质降解在细胞和发育生物学中是必不可少的，它允许细胞和组织在细胞分裂、转录、信号转导和免疫反应过程中再生和降解受损和调节的蛋白质。真核生物基因组中有超过3%的基因参与泛素介导的蛋白降解。蛋白质降解调控的失调可能导致癌症、代谢综合征、衰老和神经退行性疾病。我们想要研究一种介导泛素介导的蛋白质分解的细胞酶，称为后期促进复合体/环体(APC/C)，了解APC/C如何在细胞分裂的不同时间泛素化不同的蛋白质，以确保及时和忠实的分裂，产生两个基因相同的子细胞。这一过程的放松会导致基因不稳定，这是癌症的一个标志。因此，揭示APC/C的作用机制是癌症生物学中的一个重大突出问题。此外，APC/C调节DNA复制、DNA修复、脑功能和代谢过程，因此更好地了解APC/C在细胞中的工作方式将有利于人类健康，因为新的发现可能为早期诊断提供潜在的生物标志物，并为新药的开发提供有用的信息。目前，研究依赖APC/C的泛素化的主要困难来自于由14个亚基组成的连接酶的绝对大小和复杂性，因此对脊椎动物APC/C的非系统突变分析已有报道。现在，我们已经建立了一种利用重组的apo-APC/C结合一系列独特的生化分析方法来研究非洲爪哇APC/C的方法。在这个系统中，我们可以移除或突变不能从活细胞中删除的必需的APC/C亚基。在这个项目中，我们将重点关注Apc3和CDC20，并旨在对它们的调控进行深入的研究。该计划为新的APC/C研究提供了一个强大的平台，促进了对共激活子结合、激活和泛素化催化的详细研究，同时也帮助了翻译和临床应用的可能扩展。

Ubiquitin-mediated proteolysis is essential within cell and developmental biologyallowing cell and tissue regeneration and degradation of damaged and regulatoryproteins during cell division, transcription, signal transduction and the immuneresponse. Over 3% of genes in eukaryotic genomes are involved in ubiquitinmediatedproteolysis. Misregulation of proteolytic control can lead to cancer,metabolic syndromes, ageing and neurodegenerative disease.We want to study one of cellular enzymes that mediate the ubiquitin-mediatedproteolysis, called the anaphase-promoting complex/cyclosome (APC/C) andunderstand how the APC/C ubiquitylates different proteins at different times duringcell division to ensure the timely and faithful division to produce two geneticallyidentical daughter cells. Deregulation of this process causes genetic instability, whichis a hallmark of cancer. Thus, unravelling the mechanistic action of the APC/C is oneof the major outstanding questions in cancer biology. Furthermore, the APC/Cmodulates DNA replication, DNA repair, brain function and metabolic processes andthus a better understanding of how the APC/C works in cells will benefit for humanhealth as new finding might provide potential biomarkers useful for early diagnosisand information useful for the development of new drugs.At present, major difficulties in studying APC/C-dependent ubiquitylation arise fromthe sheer size and complexity of the ligase composed of 14 subunits and thus nosystematic mutational analysis of vertebrate APC/C has been reported. Now we havedeveloped a method to study Xenopus APC/C by reconstituted apo-APC/C togetherwith a series of unique biochemical assays. In this system, we can remove or mutateessential APC/C subunits, which cannot be deleted from living cells. In this project,we will focus on Apc3 and Cdc20 and aim to conduct an in-depth investigation oftheir regulation. This programme provides a robust platform for new APC/C research,by facilitating the detailed study of co-activator binding, activation and ubiquitylationcatalysis, while also assisting possible extension of translational and clinicalapplications.