Defining mechanisms and function of protein UFMylation

维持一个功能蛋白质组对于细胞和生物的动态平衡是必不可少的。泛素和泛素样修饰物(UBL)对蛋白质的翻译后修饰是参与许多细胞过程的信号。一个关键的作用是确保异常蛋白质被及时检测和清除，以防止错误折叠的蛋白质积累，从而维持蛋白质的动态平衡。未能清除错误折叠的蛋白质会导致有毒蛋白质聚集体的积累，这是阿尔茨海默氏症和亨廷顿病等神经退行性疾病的特征。此外，泛素和UBL在调节从DNA损伤反应和细胞应激到免疫信号的各种细胞过程中具有关键功能。UFM1是一种UBL，采用类似于泛素的β-GRAP折叠，在所有后生动物中普遍表达。重要的是，UFM1可以通过E1(UBA5)、E2(UFC1)和E3(UFL1)酶的级联反应共价连接到蛋白质的赖氨酸上，这一过程被称为UFM化。作为一种PTM，UFM化是可逆的，这是由人体内的单一酶UFSP2介导的。然而，我们仍然发现在缺乏UFSP2的细胞中存在去UFMylating活性，这表明存在一种迄今未知的UFM1蛋白酶。虽然UFM1在几个组织中普遍表达，但我们发现UFM1在B细胞准备分泌可溶性免疫球蛋白的阶段上调B淋巴细胞的激活，提示UFM化在分泌途径中发挥作用。UFM化途径组件对于小鼠的大脑发育和造血是必不可少的，因为基因切割该途径的任何组件都会导致胚胎死亡。此外，UFM化途径中的突变与一系列人类疾病有关，因此更详细地了解这种翻译后修饰是重要的。然而，我们对UFM1连接到底物上的机制、UFM1修饰的细胞蛋白的特性以及这种修饰是如何调控的了解很少。在这里，我们认为UFM1在细胞生物学中可能具有比目前所认识到的更广泛的影响。因此，我们建议的主要目的是在分子水平上了解UFM1是如何连接到底物上的，并定义UFM1修饰的不同细胞蛋白。我们还旨在发现神秘的UFM1蛋白酶在细胞中的身份，并表征其在细胞中的机制和功能。我们期待着我们的工作为UFM化提供重要的机械见解，这将加速这一新兴领域的研究。我们的工作可能有助于更好地理解UFM1如何调控分泌蛋白的生物发生和质量控制，甚至可以发现UFM化尚未相关的细胞生物学的新方面。

Maintaining a functional proteome is essential for cellular and organismal homeostasis. Posttranslational modification of proteins with ubiquitin and ubiquitin-like-modifiers (UBLs) serves as a signal involved in many cellular processes. One key role is to ensure that aberrant proteins are detected and cleared in a timely manner to prevent accumulation of misfolded proteins, thereby maintaining protein homeostasis. Failure to clear misfolded proteins results in the accumulation of toxic protein aggregates, which is characteristic of neurodegenerative disorders such as Alzheimer's and Huntington's disease. Moreover, ubiquitin and UBLs have key functions in regulating diverse cellular processes ranging from regulation of DNA damage response and cellular stress to immune signalling. UFM1 is a UBL that adopts a beta-grasp fold similar to ubiquitin and is expressed ubiquitously in all metazoans. Importantly, UFM1 can be covalently attached to lysines of proteins by an enzymatic cascade of E1 (UBA5), E2 (UFC1) and E3 (UFL1) enzymes, a process referred to as UFMylation. Being a PTM, UFMylation is reversible and this is mediated by a single enzyme UFSP2 in humans. However, we still find deUFMylating activity in cells lacking UFSP2 suggesting the presence of a hitherto unknown UFM1 protease. While ubiquitously expressed in several tissues, we find that UFM1 is upregulated upon B lymphocyte activation at the stage when B cells are poised to secrete soluble immunoglobulin, suggesting roles for UFMylation in the secretory pathway. UFMylation pathway components are essential for brain development and hematopoiesis in mice as genetic ablation of any component of the pathway results in embryonic lethality. Further, mutations in the UFMylation pathway are linked to a range of human diseases, making it important to understand this posttranslational modification in greater detail. However, we have a poor understanding of the mechanisms by which UFM1 is ligated onto substrates, the identity of the cellular proteins modified with UFM1 and how this modification is regulated. Here, we suggest that UFM1 may have far broader implications in cell biology than is currently appreciated. The main aims of our proposal are therefore to understand at the molecular level how UFM1 is ligated onto substrates and define the different cellular proteins modified with UFM1. We also aim to discover the identity of the enigmatic UFM1 protease in cells and characterize its mechanism and function in cells. We anticipate our work to provide important mechanistic insights into UFMylation, which will accelerate research in this emerging area. Our work could lead to greater understanding of how UFM1 regulates biogenesis and quality control of secretory proteins, or even uncover new aspects of cell biology that UFMylation has not been associated with.