Fig. 5. Summary of regulatory checkpoints in human MLKL activation.

Necroptosis is initiated by ligand binding to cell surface death receptors, such as TNF receptor 1, when the IAP E3 ubiquitin ligases and Caspase-8 are depleted or inhibited. Immunoprecipitation using Mb32 identified MLKL to exist in complex with RIPK3 prior to initiation of necroptosis. The RIPK3:MLKL complex is recruited to a RIPK1-nucleated platform (the necrosome) following induction of necroptosis, where RIPK3 is autophosphorylated and MLKL phosphorylated by RIPK3. Subsequently, oligomeric phosphorylated MLKL (pMLKL) disengages from RIPK3, which permits its recognition by Mb27. Phospho-MLKL oligomers are subsequently trafficked to the plasma membrane³¹ via the Golgi-/actin-/microtubule-trafficking machinery³². pMLKL accumulates at the plasma membrane into higher order hotspots, and when a threshold amount of MLKL is surpassed, MLKL ruptures the plasma membrane to induce cell death and release of pro-inflammatory DAMPs³². Monobody-27 and Monobody-32 are depicted in light and dark purple colors, respectively; dashed lines represent the interactions observed for Mb27 and Mb32 in HT29 cells. The skull and crossbones image (Mycomorphbox_Deadly.png; artist, Sven Manguard) was sourced under a Creative Commons Attribution-Share Alike 4.0 license.