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Necroptosis is a programmed form of necrosis that is dependent on activation of receptor-interacting kinase 3 (RIPK3)1 and the mixed lineage kinase domain-like (MLKL) pseudokinase2. This form of cell death is morphologically distinct from apoptosis, involving membrane rupture and release of cytoplasmic contents.
Necroptosis is activated in response to death receptor activation, although some death receptor-independent pathways are also a trigger. In most contexts, necroptosis is inhibited by proapoptotic caspase 83–5; certain intracellular pathogens suppress apoptosis by inhibiting caspase 8, and necroptosis plays a role as a back-up to eliminate infected cells6.
Necroptosis has been linked to inflammatory conditions such as multiple sclerosis7, amyotrophic lateral sclerosis (ALS), ischemia-reperfusion injury and Crohn’s disease, although it is unknown whether necroptosis is a driving factor for these diseases or a secondary consequence8.
Death receptor dependent pathway
Figure 1. Necroptosis signaling: the intersection of prosurvival and apoptotic pathways.
Necroptosis is initiated by ligand binding to death receptors including tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and Fas9,10, although TNFR1-mediated necroptosis is the most studied. These death receptors are also involved in prosurvival signaling and apoptosis: the path the cell takes is dictated by which complex forms as a result of ligand binding (Figure 1).
Upon ligand binding, the cytosolic death domain of TNFR1 recruits prosurvival complex I, consisting of TNF-receptor-associated death domain (TRADD), RIPK1 and several ubiquin E3 ligases. In complex I, RIPK1 is polyubiquitinated; however, subsequent RIPK1 deubiquitination causes RIPK1 dissociation and formation of one of two complexes: complex IIa mediates the activation of caspase 8 and initiates apoptosis, whereas complex IIb – otherwise known as the necrosome – assembles when caspase 8 is inhibited and triggers necroptosis1.
The necrosome and downstream signaling
In complex IIb, RIPK1 recruits RIPK3 through rip homeotypic interaction motifs (RHIMs) contained within both proteins, resulting in their mutual phosphorylation. RIPK3 phosphorylation results in RIPK3 oligomerization, which is necessary for its activation. Once activated, RIPK3 phosphorylates MLKL at threonine 357 and serine 3582.
Phosphorylated MLKL multimerizes and locates to the plasma membrane – a process that is crucial for necroptotic cell death11. It is currently uncertain exactly how MLKL triggers cell death12; however, there are some reports that it binds to phosphatidylinositol lipids and cardiolipin to directly permeabilize the membrane13,14.
Death receptor-independent pathway
Induction of necroptosis can also occur independently of death receptor pathways through engagement of Toll-like receptor TLR3 and TLR415, viral infection, type 1 and 2 interferons and viral expression of RHIM-containing proteins.
These proteins are part of the innate immune system and sense cellular stress, damage and infection. Upon activation, the TIR-domain-containing adapter-inducing interferon-β (TRIF) adaptor protein forms a complex with RIPK315. TLR activation-dependent necroptosis is dependent on RIPK3 and MLKL, but can proceed without RIPK1.
RIPK3-dependent necroptosis is also stimulated by activation of DAI (DNA-dependent activator of IFN regulatory factors). DAI recognizes double-strand viral DNA, and contains a RHIM domain to recruit RIPK3 and form the necrosome.
Figure 2. Pathway of viral DNA activation of necroptosis
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