T lymphocytes are key cells of our immune system that protect us against pathogens and malignant cells. T cell activation and differentiation are tightly controlled processes and deregulation can result in lymphomas, autoimmunity and inflammation. The overall aim of RELYUBL was to get a better understanding how T cell biology is regulated by ubiquitin and ubiquitin-like modifiers (UBLs). To achieve these ambitious goals, we developed novel tools and methodologies to probe ubiquitin and UBLs which include the development of selective binders, specific monoclonal antibodies and pipeline to profile deubiquitinating enzymes (DUBs) and UBL proteases in T cells. Using activity-based probes, we discovered a completely new class of deubiquitinating enzymes when we identified ZUFSP/ZUP1. We determined the crystal structure of ZUP1 which revealed a completely distinct fold that is unlike any of the known human deubiquitinating enzymes. ZUP1 is highly selective at cleaving K63-linked polyubiquitin and is important for maintaining genome stability (Kwasna et al 2018). Further, we profiled T cells for DUBs and focussed our efforts on defining the roles of OTUD6B, a DUB that is one of the most highly upregulated during T cell activation. Using a mouse model to deplete this enzyme only in T cells, our analyses reveal that OTUD6B is dispensable for T cell development but plays an important role in the differentiation of T cells to effector and memory cells.
A key area of our research explored a poorly understood UBL called UFM1. UFM1 has a similar beta-grasp fold as ubiquitin and has many parallels to ubiquitylation. However, the cellular targets and functions are not understood. Using in vitro reconstitution approaches, biochemistry, structural biology and proteomics approaches we have defined how UFM1 regulates T cell function and immune responses. Our reconstitution approaches have defined the minimal requirements for UFMylation and identified the proteins UFL1 and UFBP1 to together form a functional E3 ligase complex. Further, we identify a scaffold protein CDK5RAP3 that binds to and functions as a substrate adaptor to direct the activity of the ligase complex to endoplasmic reticulum associated ribosomes. As Ufm1 knockout mice are embryonic lethal, we have developed a model where UFM1 is deleted only in T cells or B cells. This reveals that mice lacking Ufm1 exhibit defective immune responses to infection. Applying the tools developed in Aims 1 and 2 together with proteomics and cell signalling analyses we have defined roles for UFMylation in supporting the increased secretory protein biogenesis in activated T cells. Further, we also identified how UFMylation is regulated by the two proteases UFSP1 and UFSP2. The exciting results and novel paradigms have been disseminated by peer-reviewed publications, preprints and presentations at national and international conferences.
