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Mixed lineage kinase domain-like (MLKL) is a pseudokinase containing a protein kinase-like domain that plays a critical role in tumor necrosis factor (TNF)-induced necroptosis as a functional substrate of receptor-interacting protein kinase 3 (RIP3).
Together with receptor-interacting protein 1 (RIP1), RIP3 and MLKL form a necrosis signaling complex termed the necrosome, within which MLKL is phosphorylated at T357 and S358 residues by RIP31. Once phosphorylated, MLKL oligomers localize to the plasma membrane and form disrupting pores, capable of regulating the influx of Na+ and Ca2+, and rupturing the membrane2–4.
Regulated necrosis, or necroptosis, is a newly discovered pathway, induced by mediators such as death receptors, toll-like receptors, interferons, and nucleic acid sensors. Necroptosis requires RIP3 and MLKL and is implicated in the pathogenesis of various diseases5. MLKL has attracted a great deal of interest because at this time its only known function in as a mediator of necroptosis6.
Although many of the downstream signaling components of necroptosis are unknown, MLKL and necroptosis play a role in cancer, inflammatory autoimmune, cardiovascular and neurological diseases 5,7–10. High levels of MLKL and RIP3 are associated with intestinal inflammation in children with inflammatory bowel disease (IBD)11, whereas MLKL knockdown and knockout (KO) models result in a resistance to necroptosis and localized inflammation12–14.
In multiple sclerosis (MS), the defective activation of caspase-8 and activation of RIP1, RIP3, and MLKL lead to necroptosis and mediate the degeneration of oligodendrocytes15. MS patients also show increased levels of phospho-MLKL in pathological samples compared to controls15.
However, the exact mechanism of MLKL-mediated necroptosis, the regulation of RIP3, and precisely how necroptosis contributes to disease pathogenesis remain unknown. Antibody markers against MLKL that can differentiate between unmodified and phosphorylated states will therefore be invaluable for answering these questions. A monoclonal antibody against the phosphorylated form of MLKL has already provided valuable insight into the involvement of necroptosis in drug-induced liver injury4.
The implication of necroptosis in inflammatory, cardiovascular and neurological diseases make MLKL an interesting target for the development of therapeutics. For example, modulating the activity of MLKL’s pseudokinase domain with novel small molecules could offer a means of promoting or inhibiting necroptosis under certain conditions.
The development of specific and potent MLKL modulators rests upon elucidating a more complete understanding of MLKL's activity within the context of necroptosis. This progress will only be possible with the use of highly validated and specific antibodies.
Our KO-validated MLKL antibody
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1. Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).
2. Galluzzi, L., Kepp, O. & Kroemer, G. MLKL regulates necrotic plasma membrane permeabilization. Cell Res. 24, 139–40 (2014).
3. Fuchs, Y. & Steller, H. Live to die another way: modes of programmed cell death and the signals emanating from dying cells. Nat. Rev. Mol. Cell Biol. 16, 329–344 (2015).
4. 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).
5. Pasparakis, M. & Vandenabeele, P. Necroptosis and its role in inflammation. Nature 517, 311–320 (2015).
6. Czabotar, P. E. & Murphy, J. M. A tale of two domains - a structural perspective of the pseudokinase, MLKL. FEBS J. 282, 4268–4278 (2015).
7. Brenner, D., Blaser, H. & Mak, T. W. Regulation of tumour necrosis factor signalling: live or let die. Nat. Rev. Immunol. 15, 362–374 (2015).
8. Degterev, A. et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1, 112–119 (2005).
9. Luedde, M. et al. RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling aftermyocardial infarction. Cardiovasc. Res. 103, 206–216 (2014).
10. Rickard, J. A. et al. RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis. Cell 157, 1175–1188 (2014).
11. Pierdomenico, M. et al. Necroptosis is active in children with inflammatory bowel disease and contributes to heighten intestinal inflammation. Am. J. Gastroenterol. 109, 279–87 (2014).
12. Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39, 443–453 (2013).
13. Wu, J. et al. Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res. 23, 994–1006 (2013).
14. Dannappel, M. et al. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature 513, 90–4 (2014).
15. Ofengeim, D. et al. Activation of Necroptosis in Multiple Sclerosis. CellReports 10, 1–14 (2015).