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Systematic Decoding of Deubiquitylase-Regulated Signaling Networks

Periodic Reporting for period 3 - DUB-DECODE (Systematic Decoding of Deubiquitylase-Regulated Signaling Networks)

Reporting period: 2018-10-01 to 2020-03-31

Proteins are the fundamental building blocks and major workhorses in the cell. Also, dysregulation of protein function is directly involved in diverse human diseases; therefore, understanding how proteins function in the cell and how they are regulated is vitally important. Functions of proteins critically depend on their interaction with other protein in the cell. Additionally, almost all eukaryotic proteins are regulated by diverse posttranslational modifications (PTMs) such as phosphorylation and ubiquitylation. Ubiquitylation involves conjugation of the small protein ubiquitin to other proteins though the step-wise activities of E1, E2 and E3 ubiquitin conjugating enzymes. Ubiquitylation is reversed by ubiquitin-specific proteases, called deubiquitylases (DUBs). Given the important role of ubiquitylation in many biological functions DUBs are considered attractive therapeutic candidates for targeting different diseases, such as cancer. The overall goal of the DUB-DECODE project is perform systematic studies to identify novel roles of ubiquitylation and deubiquitylation in cellular regulation. The project will help in identifying new regulatory functions of ubiquitylation and DUBs in the cell, which in turn will contribute to better understanding the role of ubiquitylation in physiology and disease.
In one of our recently published paper, we identified a novel mechanism by which the deubiquitylase CYLD is recruited to the tumor necrosis factor alpha (TNFa) receptor. TNFa is a key cytokine that functions in regulating innate immunity and inflammation. Binding of TNFa to its receptor can result in two strikingly different outcomes: activation of pro-survival signaling, or signaling that lead to cell death. CYLD is implicated as a key regulator of these decisions. We showed that the novel protein SPATA2 constitutively interacts with CYLD and recruits it to TNFa receptor; thus, SPATA2 serves a key role in determining the outcomes of TNFa receptor signaling (Wagner et al., EMBO J 2016).

Ubiquitylation-dependent protein degradation is a key regulator of eukaryotic cell division. In particular, the multiprotein ubiquitin ligase anaphase promoting complex (APC/C) is a key regulator of mitosis. While investigating the role of ubiquitylation-regulating enzymes, we found that the APC/C uses three different E2 ubiquitin-conjugating enzymes, and the concerted action of these E2s power the APC/C activity. We further discovered that it is the strength of the APC/C that makes the SAC apparatus essential in human cells and SAC becomes unessential in human cells with weakened APC/C activity (Wild et al., Cell Reports 2018, and BioRvix 2018 (pre-print)).

Protein-protein interactions are fundamental for regulating all biological processes. Therefore, identifying interaction networks of endogenously expressed proteins in highly informative. By comparing different protein-protein interaction approaches in this project, we developed a new method to investigate interaction networks of endogenously expressed proteins. As a proof of concept, we used this method to identify interaction networks in DNA damage repair signaling (Gupta et al., Cell 2018).