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

蛋白质水解是蛋白质分解成氨基酸的过程，这一过程不仅为新蛋白质合成提供了基础，而且在细胞分裂、转录、信号转导和免疫反应过程中允许细胞和组织再生和降解受损和调节蛋白。这种蛋白质水解必须是选择性的，并通过用一种称为泛素的小分子标记目标蛋白质来实现。我们体内有超过700个基因参与泛素介导的蛋白质水解，蛋白质水解控制的失调会导致癌症、代谢综合征、神经退行性疾病和衰老。在我们的实验室中，我们研究了参与这种泛素介导的蛋白质水解的关键细胞酶之一，后期促进复合物/环体（APC/C）。这种酶控制基因组DNA的复制和向子细胞的分布，以及细胞的生长、分化和死亡。此外，APC/C调节DNA损伤修复、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对人类健康有益，为发现潜在的生物标志物以早期诊断疾病提供了可能，并为开发新药提供了有用的宝贵信息。虽然APC/C的发现已经过去了20年，但我们对APC/C细胞机制的机制和控制的了解却非常有限。这部分是由于这种酶的巨大（超过13个亚基）和复杂性。此外，APC/C的许多亚基在多个位点通过添加磷酸基团（磷酸化）进行化学修饰，这是由蛋白激酶引起的。对这种化学修饰的调控知之甚少，但我们最近发现了APC/C亚基磷酸化的新机制，我们的目标是研究这种机制的详细潜在工作原理。特别是，我们将重点关注最大的亚基Apc1，并揭示Apc1磷酸化是如何被控制的，以及这如何影响APC/C在维持健康细胞中的功能。我们已经开发了一种重组APC/C酶的方法，以及一种利用爪蟾卵提取物和裂变酵母遗传学检查APC/C功能的系统。因此，我们认为该项目为新的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。为了取得最大的成果，我们将使用涉及生物化学，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。

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.