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Systematic Decoding of Deubiquitylase-Regulated Signaling Networks

Periodic Reporting for period 4 - DUB-DECODE (Systematic Decoding of Deubiquitylase-Regulated Signaling Networks)

Reporting period: 2020-04-01 to 2021-03-31

Proteins are the building blocks and main functional units of a cell. Because all living systems respond dynamically to changing environments, the stability, function, and cellular localization of proteins are dynamically controlled. There are two major mechanisms that dynamically regulate protein function in the cell: (1) interaction with other proteins, and (2) conjugation of proteins with small modifications that are known as posttranslational modifications (PTMs). Both of these mechanisms are highly prevalent and impact virtually all proteins in a cell.

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.
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.
The results obtained from the DUB-DECODE project provide important new insights into the organization of ubiquitylation signaling networks, reveal notable mechanistic insights into how ubiquitylation affects the function of selected proteins, and open new questions for future research. The technologies developed in DUB-DECODE are applicable to other areas of biology and we anticipate that they will be useful in studying other diverse aspects of biological mechanisms.
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