Structural and mechanistic regulation of phosphorylation-dependent APC/C activation

磷酸化依赖性 APC/C 激活的结构和机制调控

• 批准号: BB/N008383/1
• 负责人: Hiro Yamano
• 金额: $ 55.07万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN008383%2F1
• 关键词: Structural; mechanistic; regulation; phosphorylation; dependent

Proteolysis is the breakdown of proteins into amino acids, which is an essential process not only providing building blocks for new protein synthesis but also allowing cell and tissue regeneration and degradation of damaged and regulatory proteins during cell division, transcription, signal transduction and the immune response. This proteolysis must be selective and is achieved by tagging the target protein with a small molecule called ubiquitin. Over 700 genes in our body are involved in ubiquitin-mediated proteolysis and misregulation of proteolytic control can lead to cancer, metabolic syndromes, neurodegenerative disease and ageing.In our laboratory we study one of the key cellular enzymes involved in this ubiquitin-mediated proteolysis, the anaphase-promoting complex/cyclosome (APC/C). This enzyme controls genome DNA duplication and distribution to daughter cells as well as cell growth, differentiation and death. In addition, the APC/C modulates DNA damage repair, brain function and metabolic processes. As such, a better understanding of how the APC/C works in cells is of benefit for human health providing the possibility of discovering potential biomarkers for early diagnosis of disease and valuable information useful for the development of new drugs. Although 20 years have passed since its discovery, our understanding of the mechanism and control of the APC/C cellular machinery is lamentably limited. This is partly due to the enormity (more than 13 subunits) and complexity of the enzyme. Moreover, many subunits of the APC/C are chemically modified at multiple sites by the addition of phosphate groups ('phosphorylation') brought about by protein kinases. Very little is known about the regulation of this chemical modification but we have recently discovered a novel mechanism of phosphorylation within APC/C subunits, and we aim to investigate the detailed underlying workings of this mechanism. In particular, we will focus on the largest subunit, Apc1 and uncover how Apc1 phosphorylation is controlled and how this impacts on the function of APC/C in the maintenance of healthy cells. We have already developed a method to reconstitute recombinant APC/C enzyme as well as a system to scrutinise the function of the APC/C using Xenopus egg extracts and fission yeast genetics. Therefore, we feel this programme provides a robust platform for new APC/C research, facilitating the detailed study of phosphorylation-mediated APC/C activation via Apc1 while also giving us the opportunity to apply our findings to possible translational and clinical applications. In order to achieve a maximum outcome, we will study APC/C regulation using a multi-disciplinary approach involving biochemistry, genetics, structural biology and cell biology.

蛋白水解是将蛋白质分解为氨基酸，这是一个重要的过程，不仅为新蛋白质合成提供基础材料，而且还允许细胞和组织再生以及在细胞分裂、转录、信号转导和免疫反应过程中受损和调节蛋白质的降解。这种蛋白水解必须是选择性的，并且是通过用一种称为泛素的小分子标记靶蛋白来实现的。我们体内有超过 700 个基因参与泛素介导的蛋白水解，而蛋白水解控制的失调可导致癌症、代谢综合征、神经退行性疾病和衰老。在我们的实验室中，我们研究参与这种泛素介导的蛋白水解的关键细胞酶之一，即后期促进复合物/环体 (APC/C)。该酶控制基因组 DNA 复制和向子细胞的分配以及细胞生长、分化和死亡。此外，APC/C 还调节 DNA 损伤修复、大脑功能和代谢过程。因此，更好地了解 APC/C 在细胞中的工作原理有利于人类健康，为发现疾病早期诊断的潜在生物标志物和开发新药的宝贵信息提供了可能性。尽管自发现以来已经过去了 20 年，但令人遗憾的是，我们对 APC/C 细胞机器的机制和控制的理解仍然有限。这部分是由于酶的巨大（超过 13 个亚基）和复杂性。此外，APC/C 的许多亚基通过添加由蛋白激酶引起的磷酸基团（“磷酸化”）在多个位点进行化学修饰。人们对这种化学修饰的调节知之甚少，但我们最近发现了 APC/C 亚基内磷酸化的新机制，我们的目标是研究该机制的详细潜在工作原理。我们将特别关注最大的亚基 Apc1，并揭示 Apc1 磷酸化是如何控制的，以及它如何影响 APC/C 在维持健康细胞中的功能。我们已经开发了一种重组重组 APC/C 酶的方法，以及使用非洲爪蟾卵提取物和裂殖酵母遗传学检查 APC/C 功能的系统。因此，我们认为该计划为新的 APC/C 研究提供了一个强大的平台，促进了通过 Apc1 磷酸化介导的 APC/C 激活的详细研究，同时也使我们有机会将我们的发现应用于可能的转化和临床应用。为了取得最大成果，我们将采用涉及生物化学、遗传学、结构生物学和细胞生物学的多学科方法来研究 APC/C 调控。