Assessing the physiological relevance and molecular mechanisms of non-canonical ubiquitylation

泛素是一种小而有影响力的蛋白质，它在称为泛素化的过程中与其他蛋白质连接。泛素化是维持细胞稳态的基础。衰老或其他破坏泛素化过程的环境或遗传损伤通常导致衰弱性人类疾病的发展，包括癌症，神经变性和免疫紊乱。泛素化机制受到三种酶的严格调控，这三种酶在酶级联反应中依次起作用。在第一步中，E1酶激活泛素使其对化学敏感。然后将活化的泛素转移至E2酶（缀合酶），E2酶将其传递至E3连接酶。然后E3酶将泛素分子连接到正确的靶蛋白上。在过去的十年中，科学界已经在很大程度上相信，E3酶只能将泛素转移到一种特定类型的氨基酸，称为赖氨酸。泛素与赖氨酸的连接是通过一种称为“异肽键”的化学键发生的。这种类型的键具有很强的化学性质，非常稳定，易于检测。泛素与其他氨基酸，如丝氨酸、苏氨酸或半胱氨酸的结合被认为是罕见的例外。当泛素与丝氨酸或苏氨酸连接时，它们之间的连接，即所谓的“酯键”，比在赖氨酸上发现的“异肽”键脆弱得多。这些酯键在温和的碱性条件下很容易水解，因此被认为不够稳定，不能在真核细胞中发挥功能性作用。然而，MRC-PPU中的两个突出的小组最近发现，两种真核E3连接酶，MYCBP 2和HOIL 1，特异性地泛素化丝氨酸和苏氨酸（非典型泛素化），因此打破了“仅赖氨酸”泛素化的教条。这些研究有力地支持了丝氨酸和苏氨酸泛素化确实发生在细胞中的观点，并且由于使用了不适当的实验程序，迄今为止，非经典泛素化的证据被广泛错过。在过去的几年里，新的，高效的和高通量的技术的发展，使生物学的新方面的研究，并改变，甚至建立了新的研究领域。例如，质谱（MS）磷酸化蛋白质组学的发展极大地扩展了我们对激酶和磷酸化对细胞稳态和疾病的作用的认识。MS是一种非常重要的分析技术，允许具有这种专业知识的研究人员检测和定量蛋白质及其修饰，包括泛素化。该项目的一个重要目的是开发能够特异性鉴定和定量丝氨酸和苏氨酸泛素化的MS标准程序。这将使我们能够确定非典型泛素化在细胞中发生的程度及其生理作用。通过使用一种特殊类型的质谱仪命名为基质辅助激光解吸/电离飞行时间MS（MALDI-TOF MS），我的目的是量化和表征的E2共轭酶和E3连接酶，参与非典型泛素化的活性。我将利用我在MALDI-TOF MS方面的专业知识来回答重要的生物学问题，但也会进入不太熟悉的研究领域，如结构和化学生物学。特别是，我的目标是确定基础的分子机制，决定E2共轭酶的特异性，对丝氨酸和苏氨酸，采用结构生物学工具。我还将应用化学生物学工具来鉴定与非经典E2结合酶协同工作的E3连接酶。这个研究项目的总体愿景是揭示一种新的不同类型的泛素化。这将扩大我们对泛素系统的理解，并增加我们生产能够恢复细胞健康/稳态的药物和治疗的机会。

Ubiquitin is a small, yet influential, protein that is attached to other proteins in a process termed ubiquitylation. Ubiquitylation is fundamental in maintaining cellular homeostasis. Ageing or other environmental or genetic insults that disrupt the ubiquitylation process often lead to the development of debilitating human diseases, including cancer, neurodegeneration and immunity disorders. The ubiquitylation machinery is tightly regulated by three enzymes that act sequentially in an enzymatic cascade. In a first step, E1 enzymes activate ubiquitin to render it chemically susceptible. Activated ubiquitin is then transferred to E2 enzymes (conjugating enzymes) which pass it on to the E3 ligase enzymes. The E3 enzymes then attach the ubiquitin molecule to the correct target protein. In the last decade, the scientific community has been largely convinced that E3 enzymes were only able to transfer ubiquitin to a specific type of amino acid, called lysine. The attachment of ubiquitin to lysine happens through a chemical linkage called "isopeptide bond". This type of bond is chemically strong, very stable and easy to detect. Attachment of ubiquitin to other amino acids, such as serine, threonine or cysteine, was considered a rare exception. When ubiquitin is connected to serine or threonine, the linkage between them, which is named "ester bond", is much more fragile than the "isopeptide" bond found on lysine redisues. These ester bonds can be easily hydrolysed in mild basic conditions and therefore were considered not stable enough to have a functional role in eukaryotic cells. However, two prominent groups within the MRC-PPU recently discovered that two eukaryotic E3 ligases, MYCBP2 and HOIL1, specifically ubiquitylate serine and threonine (non-canonical ubiquitylation), therefore breaking the "lysine only" ubiquitylation dogma. These studies strongly support the idea that serine and threonine ubiquitylation is indeed happening in cells and that the evidence for non-canonical ubiquitylation has been widely missed so far because of the use of inappropriate experimental procedures. In the past years, the development of new, efficient and high-throughput technologies has enabled the study of new aspects of biology and has transformed and even established new research areas. For example, the development of phosphoproteomics by Mass Spectrometry (MS) has immensely expanded our knowledge on kinases and the role of phosphorylation on cell homeostasis and disease. MS is a very important analytical technique that allows researchers with such expertise to detect and quantify proteins and their modifications, including ubiquitylation. An important aim of this project is to develop MS standard procedures able to specifically identify and quantify serine and threonine ubiquitylation. This will allow us to determine the extent by which non-canonical ubiquitylation happens in cells as well as its physiological role. By using a particular type of Mass Spectrometer named Matrix Assisted Laser Desorption/Ionization-Time of Flight MS (MALDI-TOF MS), I aim to quantify and characterize the activity of E2 conjugating enzymes and E3 ligases that are involved in non-canonical ubiquitylation. I will exploit my expertise in MALDI-TOF MS to answer important biological questions but also move into less familiar research areas, as for structural and chemical biology. In particular, I aim to define the underpinning molecular mechanisms that determine E2 conjugating enzyme specificity toward serine and threonine by employing structural biology tools. I will also apply chemical biology tools for the identification of E3 ligases that work in tandem with non-canonical E2 conjugating enzymes. The overarching vision of this research project is to shed light on a new and different type of ubiquitylation. This will expand our understanding of the ubiquitin system and increase our chances to produce drugs and treatments able to restore cellular health/homeostasis.