Periodic Reporting for period 4 - RELYUBL (Regulation of lymphocyte biology by ubiquitin and ubiquitin like modifiers) Période du rapport: 2020-12-01 au 2021-12-31 Résumé du contexte et des objectifs généraux du projet T lymphocytes are key cells of the adaptive 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. In this research, we hypothesize that there are crucial roles undiscovered in T cells for other posttranslational modifications (PTMs) such as ubiquitin (Ub) and Ub-like proteins (UBLs). The importance of ubiquitylation in adaptive immunity is implied by the severe immunological disorders observed when components of the Ub system are disrupted in lymphocytes. It is increasingly appreciated that ubiquitin and UBL modifiers have important functions in lymphocyte biology and immunity. A major stumbling block to research in this emerging area is the complexity of the ubiquitin and UBL systems, which makes it a daunting challenge to systematically investigate these modifiers in vivo. The overall goal of RELYUBL is to better understand how T cell function and immune responses are regulated by Ub and UBL signalling networks. To achieve this goal, we developed novel tools and methodologies to study Ub and UBL modifications in cells. Using biochemical approaches we defined the molecular machinery of a poorly understood UBL called UFM1. Further, the application of activity-based probes led to the identification of novel ubiquitin proteases. By focussing on the key players identified, we have performed detailed functional analyses using innovative mouse models which have provided a better understanding of the pathways regulating T cell biology and immune responses. Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent 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. Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent) The concepts, technologies and discoveries made here will be valuable not only for immunologists but also for the entire ubiquitin research community. Very little is known about atypical polyubiquitylation and UFM1 – the modified substrates, how they are recognized, their cellular functions and how they are removed. By characterizing the enzymes involved in this posttranslational modification our research has provided mechanistic insights into how these poorly understood enzymes work. Further, our discovery of novel classes of deubiquitinating enzymes has expanded our understanding of the ubiquitin system. The elucidation of substrates of ubiquitin and UBL proteases will pave the way for future research to investigate substrate targeting and mechanisms regulating catalytic activity of these enzymes. The tools, methods and strategies we have established here for studying ubiquitin/UBL modifications in lymphocytes can be extended to other biological systems. Given the emerging interest in targeting the ubiquitin pathway for therapy, the knowledge gained in this proposal can be exploited for the development of selective inhibitors. We expect that UFM pathway components will represent potent lymphocyte-specific therapeutic targets for lymphoma and immune disorders, which will be explored beyond this project. Furthermore, I strongly believe that a deeper understanding of the ubiquitin/UBL-dependent biochemical circuitry controlling lymphocyte biology will provide novel strategies for modulating T cell differentiation to increase anti-tumour efficacy in immunotherapy.
