Necroptosis

Necroptosis is a programmed form of necrosis that is dependent on activation of receptor-interacting protein kinase 3 (RIPK3)² and the mixed lineage kinase domain-like (MLKL) pseudokinase³. 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 8^{4, 5, 6} , certain intracellular pathogens suppress apoptosis by inhibiting caspase 8, and necroptosis plays a role as a backup to eliminate infected cells⁷. Necroptosis has been linked to inflammatory conditions such as multiple sclerosis⁸, 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 consequence⁹.

Death receptor dependent pathway

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 Fas^10,11, 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 ubiquitin 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 necroptosis².

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 358³.

Phosphorylated MLKL multimerizes and locates to the plasma membrane – a process that is crucial for necroptotic cell death¹². It is currently uncertain exactly how MLKL triggers cell death¹³; however, there are some reports that it binds to phosphatidylinositol lipids and cardiolipin to directly permeabilize the membrane ^14,15.

Figure 1. Necroptosis triggered by TNFR1.

Death receptor-independent pathway

Induction of necroptosis can also occur independently of death receptor pathways through engagement of Toll-like receptor TLR3 and TLR4¹⁶, viral infection, type 1 and 2 interferons and viral expression of RHIM-containing proteins (Figure 2).

Toll-like receptors

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 RIPK3¹⁶. TLR activation-dependent necroptosis is dependent on RIPK3 and MLKL, but can proceed without RIPK1.

Viral DNA

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. Death receptor-independent pathways of necroptosis.

References

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2 . Moriwaki, K. & Chan, F. K. M. RIP3: A molecular switch for necrosis and inflammation. Genes Dev. 27, 1640–1649 (2013).

3 . Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.  Cell 148, 213–227 (2012).

4 . Donnell, M . A . O . et al . NIH Public Access. 13, 1437–1442 (2012).

5 . Lin, Y., Devin, A., Rodriguez, Y. & Liu, Z. G. Cleavage of the death domain kinase RIP by Caspase-8 prompts TNF-induced apoptosis. Genes Dev. 13, 2514–2526 (1999).

6 . Feng, S. et al. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cell. Signal. 19, 2056–2067 (2007).

7 . Mocarski, E. S., Guo, H. & Kaiser, W. J. Necroptosis: The Trojan horse in cell autonomous antiviral host defense. Virology 479–480, 160–166 (2015).

8 . Ofengeim, D. et al. Activation of necroptosis in multiple sclerosis.  Cell Rep. 10, 1836–49 (2015).

9 . Conrad, M., Angeli, J. P. F., Vandenabeele, P. & Stockwell, B. R. Regulated necrosis: disease relevance and therapeutic opportunities. Nat. Rev. Drug Discov. 15, 348–366 (2016).

10 . Degterev, A. et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.  Nat Chem Biol 1, 112–119 (2005).

11 . Holler, N. et al. Fas triggers an alternative, caspase-8–independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1, 489–495 (2000).

12 . Cai, Z. et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16, 55–65 (2014).

13 . Galluzzi, L., Kepp, O. & Kroemer, G. MLKL regulates necrotic plasma membrane permeabilization . Cell Res. 24, 139–40 (2014).

14 . Dondelinger, Y . et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates.  Cell Rep. 7, 971–81 (2014).

15 . Wang, H . et al. Mixed Lineage Kinase Domain-like Protein MLKL Causes Necrotic Membrane Disruption upon Phosphorylation by RIP3.  Mol. Cell 54, 133–146 (2014).

16 . He, S., Liang, Y., Shao, F. & Wang, X. Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway.  Proc. Natl. Acad. Sci. 108, 20054–20059 (2011).