Necroptosis is a form of regulated necrosis that results in cell death and content release after plasma membrane permeabilization. However, little is known about the molecular events responsible for the disruption of the plasma membrane. Here, we find that early increase in cytosolic calcium in TNF-induced necroptosis is mediated by treatment with a Smac mimetic via the TNF/RIP1/TAK1 survival pathway. This does not require the activation of the necrosome and is dispensable for necroptosis. Necroptosis induced by the activation of TLR3/4 pathways does not trigger early calcium flux. We also demonstrate that necroptotic plasma membrane rupture is mediated by osmotic forces and membrane pores around 4 nm in diameter. This late permeabilization step represents a hallmark in necroptosis execution that is cell and treatment independent and requires the RIP1/RIP3/MLKL core. In support of this, treatment with osmoprotectants reduces cell damage in an in vivo necroptosis model of ischemia-reperfusion injury.

Necroptosis is a recently discovered form of regulated cell death characterized by the disruption of plasma membrane integrity and the release of intracellular content. Mixed lineage kinase domain-like (MLKL) protein is the main player of this cell death pathway as it mediates the final step of plasma membrane permeabilization. Despite the significant progress in our knowledge of the necroptotic pathway and MLKL biology, the precise mechanism of how MLKL functions remain unclear. To understand in what way MLKL executes necroptosis, it is crucial to decipher how the molecular machinery of regulated cell death is activated in response to different stimuli or stressors. It is also indispensable to unveiling the structural elements of MLKL and the cellular players that are required for its regulation. In this review, we discuss the key steps that lead to MLKL activation, possible models that explain how it becomes the death executor in necroptosis, and its emerging alternative functions. We also summarize the current knowledge about the role of MLKL in human disease and provide an overview of existing strategies aimed at developing new inhibitors that target MLKL for necroptosis intervention.

Crystallographic data of the dimeric and octameric forms of fragaceatoxin C (FraC) suggested the key role of a small hydrophobic protein–protein interaction surface for actinoporins oligomerization and pore formation in membranes. However, site‐directed mutagenesis studies supporting this hypothesis for others actinoporins are still lacking. Here, we demonstrate that disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII), and sticholysin II (StII) impairs the pore formation activity of these proteins. Our results allow for the extension of the importance of FraC protein–protein interactions in the stabilization of the oligomeric intermediates of StII and EqtII pointing out that all of these proteins follow a similar pathway of membrane disruption. These findings support the hybrid pore proposal as the universal model of actinoporins pore formation. Moreover, we reinforce the relevance of dimer formation, which appears to be a functional intermediate in the assembly pathway of some different pore‐forming proteins.

Pyroptosis, necroptosis, and ferroptosis are well-characterized forms of regulated necrosis that have been associated with human diseases. During regulated necrosis, plasma membrane damage facilitates the movement of ions and molecules across the bilayer, which finally leads to cell lysis and release of intracellular content. Therefore, these types of cell death have an inflammatory phenotype. Each type of regulated necrosis is mediated by a defined machinery comprising protein and lipid molecules. Here, we discuss how the interaction and reshaping of these cellular components are essential and distinctive processes during pyroptosis, necroptosis, and ferroptosis. We point out that although the plasma membrane is the common target in regulated necrosis, different mechanisms of permeabilization have emerged depending on the cell death form. Pore formation by gasdermins (GSDMs) is a hallmark of pyroptosis, while mixed lineage kinase domain-like (MLKL) protein facilitates membrane permeabilization in necroptosis, and phospholipid peroxidation leads to membrane damage in ferroptosis. This diverse repertoire of mechanisms leading to membrane permeabilization contributes to define the specific inflammatory and immunological outcome of each type of regulated necrosis. Current efforts are focused on new therapies that target critical protein and lipid molecules on these pathways to fight human pathologies associated with inflammation.