Deconstructing the Checkpoints of Necroptosis

For a long time, apoptosis was considered the sole form of programmed cell death during development, homeostasis, and disease, whereas necrosis was regarded as an unregulated and uncontrollable process. Evidence now reveals that necrosis can also occur in a regulated manner. Programmed necrosis, 'necroptosis' plays vital roles during host-pathogen interactions where it is triggered as host-defence mechanism for the elimination of pathogen-infected cells. However, necroptosis also participates in the pathogenesis of diseases, including ischaemic injury, neuro-degeneration, and viral infection. Moreover, necroptosis has also sparked considerable interest among cancer researchers for its potential to overcome tumour resistance to apoptosis, and because it is more immunogenic than apoptosis, flagging up tumours for immunological attack. For these reasons, there has been much interest in obtaining a better understanding of how necroptosis is activated and how this potentially catastrophic event is regulated.

Necroptosis is mediated by MLKL, a membrane permeabilizing pseudo-kinase that translocates to the plasma membrane upon its activation. While necroptosis signalling has attracted much attention for its therapeutic potential, little is known how necroptosis is regulated, and how MLKL translocates to hotspots at the plasma membrane to trigger necroptosis.

We now have identified that the Ubiquitin (Ub)-signalling system critically regulates necroptosis, and that ubiquitylation of MLKL is required for MLKL to traffic to the plasma membrane. Moreover, we have identified several putative MLKL-regulatory Ub-E3 ligases and deubiquitylating enzymes that might operate as decisive necroptotic checkpoint.

Aim:
The aim of this proposal is to identify the mechanism through which active MLKL translocates to the plasma membrane where it accumulates at hotspots to cause lytic cell death. We will also identify the intercellular structures at which MLKL accumulates and characterise their contribution to necroptosis signalling and membrane rupture.

To achieve this, we will characterise the molecular players of the Ub signalling system (E3 ligases, deubiquitylating enzymes and Ub-receptors) that underpin MLKL ubiquitylation, trafficking and accumulation at intercellular contact sites. Moreover, we will study whether the identified E3 ligases, deubiquitylating enzymes and Ub-receptors contribute to antiviral host defence.

Methods:
Using biochemical, single-cell imaging and in vivo approaches, we will elucidate how MLKL is ubiquitylated by E3 ligases, how the ubiquitylation status of MLKL is edited by deubiquitylating enzymes, and how such signalling chains are detected by Ub-binding proteins (Ub-receptors) to shuttle active MLKL to intercellular hotspots at the plasma membrane. Moreover, we will unravel the role of the identified Ub-E3 ligases, deubiquitylating enzymes and Ub-receptors in modulating anti-viral host defences. Further, we will evaluate the contribution of desmosomes and Flotillins in necroptosis signalling.

How the results will be used
A better understanding of necroptosis signalling will be of enormous interest to basic scientists as well as clinical researchers because it will lay the foundation for the design of future therapeutic strategies aimed at boosting antiviral defence, fighting cancer and suppressing inflammatory diseases.

Necroptosis is a recently discovered cell death modality that is rapidly emerging as an important mediator of animal and human pathologies. Moreover, necroptosis has also sparked considerable interest among cancer researchers for its potential to overcome tumour resistance to apoptosis.

Although the initial steps of MLKL activation are quite well established, what follows between its phosphorylation by RIPK3, and its oligomerization, translocation, and insertion in the plasma membrane to execute necroptosis is still not understood. While phosphorylation of MLKL constitutes a key signalling step, additional check points exist along the trafficking routes of MLKL to the plasma membrane. However, little is known about the molecular players that control these necroptotic checkpoints.

We now have identified that the Ub-signalling system critically regulates MLKL-mediated necroptosis, and that ubiquitylation of MLKL is required for MLKL to translocate and accumulate at plasma membrane hotspots. Further, we identified several candidate E3 ligases and DUBs that might operate as decisive necroptotic checkpoints.

To learn more about trafficking-mediated regulation of necroptosis, we propose the following set of investigations:

1: Identify the players of the Ub-signalling system that traffics MLKL to membrane hotspots
2: Discover the role of desmosomes in necroptosis signalling
3: Investigate the role of the identified trafficking components in regulating anti-viral host defence

We anticipate that our work will uncover fundamental mechanisms of necroptosis regulation, creating the necessary knowhow to strengthen our defences against viral pathogens. In addition, it will provide clues how to suppress inflammatory diseases and improve anti-tumour therapy.

Therefore, our findings will have a wide-ranging impact on diverse aspects of mammalian biology.