Defining mechanisms and function of protein UFMylation

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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.