Necroptosis is a form of cell death, inhibited by the FADD-caspase-8-FLIP complex, in which the interaction of two protein kinases, RIPK1 and RIPK3, interact to promote or inhibit necrotic death of a cell. Activated RIPK3 phosphorylates the pseudokinase MLKL, which mediates necroptosis through action at the plasma membrane. We have probed the late events that surround MLKL activation and have found that prior to loss of plasma membrane integrity, cell death is reversible. We have implicated the ESCRT III pathway, which functions in endosomal trafficking, cytokinesis, and exocytosis, in counter-acting the membrane effects of active MLKL. This results in the release of ?bubbles? of damaged plasma membrane from the cell surface, sustaining cell survival. Our central hypothesis, upon which this application is based, is that necroptosis proceeds via the activation of MLKL and its action at the plasma membrane, and the regulation of this late process allows other events, such as protein expression induced by the necroptotic stimulus to manifest and thereby impact on the effects of the dying cell on the body. Based on this hypothesis, we will ask: 1. How do the late events in necroptosis regulate cell death and survival? Here we will identify the components of ESCRT III responsible for bubble formation after MLKL activation, and their role in ?resuscitation? when active MLKL is subsequently disrupted. We will determine how ESCRT III functions to inhibit necroptosis in response to inducers without caspase inhibition, and explore if RIPK3 activity, independently of MLKL, may regulate the ESCRT III pathway. 2. Are there non-canonical pathways to MLKL activation and necroptosis? While several inducers of necroptosis have been identified, we propose that there are others that are less explored. We have identified DNA damage as an inducer of necroptosis, and will explore how this occurs. Further, we have found agents that appear to activate MLKL and necroptosis independently of RIPK3, and will determine how this non-canonical activation occurs. 3. What are the consequences of the late events in necroptosis for other cells? Cells undergoing necroptosis affect surrounding cells. Here we will ask how ESCRT III impacts on engulfment of the dying cell, how the delay in death supports gene expression and protein production, and allows engagement of adaptive immunity to antigens associated with the dying cell. Completion of our goals will advance our fundamental understanding of how necroptosis is regulated, and how it can be exploited in therapies directed to cancer and other pathological conditions.
Necroptosis is a form of regulated necrotic death, in which the FADD-caspase-8-FLIP complex and RIPK1 control the activation and function of RIPK3, which in turn activates MLKL to target the plasma membrane. In this proposal we investigate the final stages of MLKL activation and necroptosis to determine how it is regulated and how this affects surrounding cells.
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