Ubiquitin-mediated regulation of necroptosis

Abstract

Regulated cell death is a fundamental cellular process that is critical for the development and survival of multicellular organisms. Cell death and inflammation are essential to restore tissue homeostasis following tissue malfunction, injury or infection. Deregulation of the signalling pathways that trigger cell death can lead to the development of diseases such as cancer or autoimmunity. Necroptosis is a type of regulated cell death characterized by the rupture of the plasma membrane and the release of the inflammatory components into the tissue microenvironment thereby triggering inflammation and immune responses. The pseudokinase MLKL constitutes the last effector of the necroptotic pathway. Currently, little is known about the mechanism of regulation of MLKL and how it mediates cell death execution. In this thesis, I studied the role of Ubiquitin (Ub) in regulating MLKL-mediated necroptosis. I discovered that MLKL is heavily ubiquitylated during necroptosis and that this contributes to its killing activity. Ubiquitylation of MLKL occurs downstream of its phosphorylation by RIPK3. Using mass spectrometry, I have identified four lysine (K) residues of MLKL that serve as ubiquitin acceptor sites. Among these, I have focused on K219 which is positioned within MLKL's pseudoactive site. Ubiquitylation at this site is important in controlling MLKL's killing potential as cells derived from MlklK219R knockin animals are less sensitive to undergo necroptosis. Mechanistically, ubiquitylation enhances the ability of phosphorylated MLKL 9P-MLKL) to form higher order oligomers at the plasma membrane. I have also investigated MLKL's interactome and its localization during necroptosis. I identified several desmosomal proteins that interact with MLKL. In line with this, I found that P-MLKL preferentially localizes to regions of the plasma membrane where cells are in contact with one another. In summary, my study describes how the Ub system modulates MLKL activity and identifies cell-cell contact points as 'hotspots' for MLKL recruitment.