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确功能。因此，我们必须彻底了解发生在它们体内的生化过程。正是这些过程在某些疾病中出现了缺陷，因此，掌握了这些疾病的复杂性，我们就可以合理地开发新的治疗方法，减轻它们的症状，甚至治愈它们。一种影响所有过程的细胞调节机制被称为蛋白质泛素化。蛋白质泛素化是指一种叫做泛素的小蛋白质附着在其他蛋白质上。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，在我们细胞中成千上万的蛋白质中，特定的蛋白质可以被泛素标记。这种特异性是由大约600种被称为E3连接酶的蛋白质酶实现的。如果E3有缺陷，并且活性降低或升高，那么这可能表现为疾病。由于缺乏能够测量细胞中单个E3活性的研究技术，全面了解蛋白质泛素化的挑战就产生了。此外，我们现在对哪些蛋白质被泛素标记有很强的了解，但我们识别E3的能力非常困难，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E3s的活性，还可以同时测量数十个E3s的活性。这项技术将彻底改变我们理解E3s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附着到特定底物上的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.