Periodic Reporting for period 4 - DUB-DECODE (Systematic Decoding of Deubiquitylase-Regulated Signaling Networks)
Période du rapport: 2020-04-01 au 2021-03-31
Among different PTMs, ubiquitylation has emerged as the main mechanism of regulating protein stability in eukaryotes. Ubiquitylated proteins are targeted to the macromolecular machine, called the proteasome. The discovery of the ubiquitin-proteasome system received the Nobel Prize in medicine in 2004. Over the past two decades, it has become clear that ubiquitylation function extends far beyond and involves processes such as cell division, replication, cell death, and signaling. Furthermore, dysregulation in ubiquitylation is directly implicated in human disease, and inhibitors targeting proteasome activity are used in the clinic. Beyond the proteasome, deubiquitylases (DUBs) have emerged as attractive therapeutic candidates for autoimmune disorders, chronic inflammation, oncology, and neurodegeneration.
Understanding how ubiquitin signaling networks are organized, and how ubiquitin regulates different biological processes, is fundamental for understanding how human cells work. It is hoped that, in the longer term, an improved understanding of ubiquitylation signaling will pave the way for developing selective therapeutic strategies that target the components of ubiquitin signaling machinery, such as DUBs. The goal of the DUB-DECODE project is to improve the understanding of ubiquitylation signaling in human cells by systematically mapping proteins that interact with human DUBs and identifying proteins that are regulated by DUBs. Also, the project aims to develop new technologies for studying ubiquitylation and protein-protein interactions. Finally, the project aims to study the functional roles of ubiquitylation. The main conclusion of the project is that ubiquitylation signaling is very rapidly regulated, and DUB activity is crucial for rapid regulation.
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.