In the DUB-DECODE project, we used genetically engineered human cells and quantitative mass spectrometry to understand the organization and function of ubiquitylation signaling networks. 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 immune and inflammatory signaling, and dysregulation of TNFa signaling is implicated in autoimmune and inflammatory diseases. Activation of TNFa receptor can result in two strikingly different outcomes: activation of signaling that promotes cell survival, or cell death. CYLD is a key regulator of these decisions. We found that SPATA2, a previously unstudied protein, constitutively interacts with CYLD and recruits it to the TNFa receptor. By doing so, SPATA2 plays 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. 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 spindle assembly checkpoint (SAC) apparatus essential in human cells, and if APC/C activity is weakened, the SAC becomes unessential for the viability of human cells (Wild et al., Cell Reports 2016, and 2018).
Protein-protein interactions are fundamental for regulating all biological processes. Therefore, studying interaction networks of endogenously expressed proteins is 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). We identified a novel protein complex, termed Shieldin, which is composed of REV7 and three previous uncharacterized proteins. Shieldin promotes DNA double-strand break repair by non-homologous end-joining, and this is crucial for generating antibody diversity through the process called antibody class-switching. Shieldin determines the sensitivity of clinically approvided PARP inhibitors to homology-directed repair-deficient cancer cells. We further showed that regulation of replication speed is important for genome maintenance (Somyajit et al., Science 2017), and identified a novel role of the MCMBP protein in preventing ubiquitylation-mediated degradation of MCM protein. We found that, before cell division, cells synthesize a large pool of MCM proteins and MCMBP protects the newly synthesized MCP proteins from degradation. The nascent and parental pool of MCM proteins are both important for maintaining the genomes of replicating cells. Parental MCM proteins preferentially mature into the productive CDC45-MCM-GINS (CMG) helicases, whereas the nascent MCM protein regulates the speed and symmetry of the replicating forks (Sedlackova et al., Nature 2020). To disseminate the findings of the project, the results were presented at several international conferences, the results are published in peer-reviewed journals and made freely accessible.
