MLKL and its Role in the Necroptosis Pathway

Mixed lineage kinase domain-like (MLKL) plays an important role in necroptosis and is also associated with several diseases in humans.

The Function of MLKL

MLKL is a pseudokinase protein that possesses a kinase-like region, which is involved in necroptosis induced by tumor necrosis factor (TNF) - where it acts 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.

Following phosphorylation oligomers of MLKL migrate to the plasma membrane of cells where they participate in the formation of disrupting pores. These pores regulate the influx of Ca2+ and Na+ into the cell, resulting in membrane rupture.2-4

Disease and MLKL

Nectroptosis is a recently discovered pathway used by cells to regulate necrosis. The pathway is regulated by mediators including nucleic acid sensors, interferons, death receptors and toll-like receptors.

For necroptosis to occur both MLKL and RIP3 must be present, and the pathway is associated with the progression of several diseases.5 MLKL is a molecule of high interest because, at the time of writing, its only known function is as a regulator of necroptosis.6

Whilst many of the downstream signaling molecules of the necroptosis pathway remain unknown, research has demonstrated that necroptosis, and therefore MLKL, is involved in cancer, cardiovascular, autoimmune and neurological diseases.5,7-10

Elevated concentrations of RIP3 and MLKL have been linked to inflammation of the intestines in children with inflammatory bowel disease (IBD)11, and MLKL knockdown and knockout (KO) models result in resistance to necroptosis and localized inflammation.12-14

During the progression of multiple sclerosis (MS), the activation of MLKL, RIP1 and RIP3, in addition to the defective activation of caspase-8, results in necroptosis and the regulation of oligodendrocyte degeneration. Samples from MS patients have also been found to exhibit higher levels of phospho-MLKL when compared to controls.15

Despite knowing that MLKL plays a key role in the process of necroptosis, the knowledge of the exact mechanism by which MLKL regulates the process, how RIP3 is regulated and the exact contribution of necroptosis towards disease progression remains unknown.

Using antibodies targeted towards MLKL allows the differentiation between phosphorylated and un-phosphorylated MLKL and will allow researchers to determine the exact relationships between these factors.

Using a monoclonal antibody targeted towards phosphorylated MLKL has already been shown to provide insight into the relationship between necroptosis and drug-induced liver injury.4

Therapy and MLKL

The association of necroptosis with diseases involving the neural system, cardiovascular system and inflammation makes MLKL a potential target for new therapies.

One potential therapy could modify the activity of the pseudokinase domain in MLKL using novel small molecules, which could allow the inhibition or promotion of necroptosis under different conditions.

Identifying and developing strong MLKL modulating small molecules requires there to be a greater understanding of the activity of MLKL and how it regulates necroptosis. Developing this understanding is only possible with the use of highly selective, validated antibodies.

Abcam’s KO-Validated MLKL Antibody

  • Validated for use in immunohistochemistry and western blot (formalin/PFA-fixed, paraffin-embedded section)
  • Recombinant rabbit monoclonal [EPR17514] anti-MLKL antibody (ab184718)
  • Antibody specificity is confirmed with a true negative control (KO validation)
  • RabMAb® technology provides improved bioactivity and stronger binding
  • Validated independently by Abcam’s customers

References

  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).

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: May 24, 2019 at 7:48 AM

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