The discovery of alternative signaling pathways that regulate cell death has revealed multiple strategies for promoting cell death with diverse consequences at the tissue and organism level. Despite the divergence in the molecular components involved, membrane permeabilization is a common theme in the execution of regulated cell death. In apoptosis, the permeabilization of the outer mitochondrial membrane by BAX and BAK releases apoptotic factors that initiate the caspase cascade and is considered the point of no return in cell death commitment. Pyroptosis and necroptosis also require the perforation of the plasma membrane at the execution step, which involves Gasdermins in pyroptosis, and MLKL in the case of necroptosis. Although BAX/BAK, Gasdermins and MLKL share certain molecular features like oligomerization, they form pores in different cellular membranes via distinct mechanisms. Here, we compare and contrast how BAX/BAK, Gasdermins, and MLKL alter membrane permeability from a structural and biophysical perspective and discuss the general principles of membrane permeabilization in the execution of regulated cell death.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBIDmediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

BFL1 is a relatively understudied member of the BCL2 protein family which has been implicated in the pathogenesis and chemoresistance of a variety of human cancers, including hematological malignancies and solid tumours. BFL1 is generally considered to have an antiapoptotic function, although its precise mode of action remains unclear. By quantitatively analyzing BFL1 action in synthetic membrane models and in cells, we found that BFL1 inhibits apoptosis through three distinct mechanisms which are similar but not identical to those of BCLXL, the paradigmatic antiapoptotic BCL2 family protein. Strikingly, alterations in lipid composition during apoptosis activate a prodeath function of BFL1 that is based on noncanonical oligomerization of the protein and breaching of the permeability barrier of the outer mitochondrial membrane (OMM). This lipid-triggered prodeath function of BFL1 is absent in BCLXL and also differs from that of the apoptotic effector BAX, which sets it apart from other BCL2 family members. Our findings support a new model in which BFL1 modulates apoptosis through a bifunctional and multimodal mode of action that is distinctly regulated by OMM lipids compared to BCLXL.