Activity-based Proteomics of E3 Ligases

我们的健康和幸福取决于我们体内细胞的正常运作。因此，我们必须彻底了解其中发生的生化过程。正是这些过程在某些疾病中变得有缺陷，因此掌握了它们复杂性的知识将使我们能够合理地开发新的疗法来缓解症状甚至治愈它们。影响所有过程的细胞调节机制被称为蛋白质泛素化。蛋白质遍在化涉及一种称为遍在蛋白的小蛋白质与其他蛋白质的附着。这种修饰是多用途的，但最重要的功能之一是标记受损或不需要的蛋白质以进行破坏。值得注意的是，在我们细胞中的1000种蛋白质中，特定的蛋白质可以用泛素标记。这种特异性是由大约600种蛋白酶（称为E3连接酶）实现的。如果E3有缺陷，并且活动减少或增加，那么这可能表现为疾病。充分理解蛋白质泛素化的挑战来自于缺乏研究技术，即使是细胞中单个E3的活性也无法测量。此外，我们现在对哪些蛋白质被泛素标记有很强的了解，但我们鉴定E3的能力是非常困难的，迫切需要新的工具。我们最近开发了一些令人兴奋和强大的技术，不仅可以测量细胞中单个E3的活性，而且可以同时测量数十个E3的活性。这项技术将彻底改变我们理解E3和蛋白质泛素化在正常和病变细胞中的作用的能力。我的研究计划的第一部分将建立和优化这项技术。该提案的第二部分是开发和优化第二种技术，这将有助于我们识别负责将泛素连接到特定底物的E3的能力。

Our health and well-being are dependent on the correct functioning of the cells in our body. It is therefore imperative that we have a thorough understanding of the biochemical processes that take place within them. It is these processes that become faulty in certain diseases therefore being armed with the knowledge of their intricacies will place us in a position where we can rationally develop new therapies that alleviate their symptoms or even cure them.A cellular regulatory mechanism that affects all processes is known as protein ubiquitination. Protein ubiquitination involves the attachment of a small protein called ubiquitin to other proteins. This modification is multipurpose but one of the most significant functions is to mark damaged or unwanted proteins for destruction. Remarkably, out of the 1000's of proteins in our cells, specific proteins can be tagged with ubiquitin. This specificity is achieved by ~600 protein enzymes known as E3 ligases. If an E3 is faulty and has reduced or elevated activity, then this can manifest itself as disease. Challenges with fully understanding protein ubiquitination arise from a lack of research technologies that enable the activities of even a single E3 in a cell to be measured. Additionally, we now have strong knowledge about which proteins are tagged with ubiquitin but our ability to identify the E3 responsible is extremely difficult and new tools are urgently needed.We have recently developed some exciting and powerful technology that allows not only a single E3s activity in a cell to be measured, but the activity of tens of E3s to be measured simultaneously. This technology will revolutionise our ability to understand the roles of E3s and protein ubiquitination in both normal and diseased cells. The first part of my research proposal will establish and optimise this technology. The second part of this proposal is to develop and optimise a second technology that will facilitate our ability to identify the E3s that are responsible for attaching ubiquitin to specific substrates.