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

泛素介导的蛋白质水解在细胞和发育生物学中是必不可少的，允许细胞和组织再生和降解受损和细胞分裂、转录、信号转导和免疫反应中的调节蛋白。真核生物基因组中超过3%的基因参与泛素介导的蛋白质水解。蛋白质水解控制的失调可导致癌症、代谢综合征、衰老和神经退行性疾病。我们希望研究一种介导泛素介导的蛋白质水解的细胞酶，称为后期促进复合物/环体（APC/C），并了解APC/C如何在细胞分裂的不同时间泛素化不同的蛋白质，以确保及时和忠实地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的机制作用是癌症生物学中主要悬而未决的问题之一。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如何在细胞中起作用将有益于人类健康，因为新的发现可能为早期诊断提供潜在的生物标志物，并为新药开发提供有用的信息。目前，研究APC/C依赖性泛素化的主要困难在于由14个亚基组成的连接酶的大小和复杂性，因此尚未有脊椎动物APC/C的系统突变分析报道。目前，我们建立了一种利用重组apo-APC/C结合一系列独特的生化检测方法来研究非洲爪蟾APC/C的方法。在这个系统中，我们可以去除或突变APC/C亚基，这些亚基不能从活细胞中删除。在这个项目中，我们将以Apc3和Cdc20为重点，对它们的调控进行深入的调查。该项目为新的APC/C研究提供了一个强大的平台，通过促进对共激活剂结合、激活和泛素化催化的详细研究，同时也有助于可能的翻译和临床应用的扩展。

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.