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Regulation of lymphocyte biology by ubiquitin and ubiquitin like modifiers

Periodic Reporting for period 2 - RELYUBL (Regulation of lymphocyte biology by ubiquitin and ubiquitin like modifiers)

Reporting period: 2017-12-01 to 2019-05-31

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. Hence, the biochemical events regulating lymphocyte biology have long been a topic of intense research, which has been focussed predominantly on protein phosphorylation. 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. These insights have mainly come from genetic and biochemical studies of candidate E3 enzymes and DUBs. However, these studies investigating single candidates do not reveal the true extent to which ubiquitylation regulates T cell biology. 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 this proposal is to better understand how T cell function and immune responses are regulated by Ub and UBL signalling networks. To achieve this goal, we are using a systematic unbiased approach to define how T-lymphocyte function and immune responses are regulated by ubiquitin and UBL-mediated signalling networks. Specifically, we aim to establish methods to probe the ubiquitin and UBL systems in vivo. Using proteomic approaches, we will define the proteins modified by ubiquitin and UBLs and study the dynamic changes during T cell activation. By focussing on the ubiquitin/UBL proteases that are expressed in T cells, we aim to understand the pathways regulating T cell biology and immune responses. Lastly, we are developing innovative in vivo models to investigate the functional importance of key players that we have identified in T cell function and immune responses following encounter of pathogen.
The overall aim of this work is to get a better understanding how T cell biology is regulated by ubiquitin and ubiquitin-like modifiers. The first aim of this project was to understand how polyubiquitin and Ubls modifications are decoded in T cells. We are establishing reconstitution systems using recombinant enzymes to assemble these different modifications. These efforts have not only led to important reagents but has also provided novel insights into the enzymatic machinery. In addition, we have been developing monoclonal antibodies to map the ubiquitin and Ubl modification during T cell activation.
To understand the regulatory networks of ubiquitin and UBL signalling, we have developed activity-based probes to profile deubiquitinating enzymes and Ubl proteases in T cells. This reveals not only the active proteases in T cells but also is an opportunity to identify novel proteases. In exciting results, we discovered a completely new class of deubiquitinating enzymes when we identified ZUFSP/Zup1 in our pull downs. While misannotated as a Ufm1-specific peptidase, this completely uncharacterized enzyme is indeed a deubiquitinating enzyme. We determined the crystal structure of Zup1 which revealed a completely distinct fold that is unlike any of the known human deubiquitinating enzymes. Further, we identified two new ubiquitin binding domains in this DUB. Zup1 is highly selective at cleaving K63-linked polyubiquitin and is important for maintaining genome stability. We published our findings in Molecular Cell (Kwasna et al). Further, we profiled T cells for DUBs that are highly upregulated during T cell activation. For detailed functional characterization, we have focussed our efforts on key enzymes. We have generated floxed mice which have been crossed to CD4-Cre mice to knockout these enzymes only in T cells. We are now analysing the functional consequence of deleting these enzymes to T cell function and immune responses. Our preliminary results suggest an important role for these highly regulated deubiquitinases in the differentiation of T cells to effector cells and in immune responses following infection.
A key area of our research is to explore a poorly understood post translational modifier 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. We are therefore using in vitro reconstitution approaches, biochemistry, structural biology and proteomics approaches to understand how Ufm1 that is highly expressed in lymphocytes regulates their function and immune responses. We are also performing detailed functional characterization of the roles of ufmylation in T cells. As Ufm1 knockout mice are embryonic lethal, we have developed a model where Ufm1 is deleted only in T cells. Using this model, we will the functional importance of this modifier in T cells following infection. These experiments are currently underway. In summary, since the start of this project, we have made significant progress and have exciting results and indications that we are studying in detail.
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 will provide 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 strategies we will establish here for identifying ubiquitin/UBL protease substrates in lymphocytes can be extended to other biological systems. Given the emerging interest in exploring this class of enzymes as therapeutic targets, we will exploit the knowledge gained in this proposal to engineer selective inhibitors. Since selective allosteric inhibitors are superior to electrophilic compounds that target the catalytic cysteine, we first need a greater understanding of cellular substrates and mechanisms of action are vital. We expect that ubiquitin and UBL proteases 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.