This technology addresses the phenomenon of necroptosis, a regulated form of cell death that has significant implications for various diseases and traumas. Specifically, it focuses on MLKL polymerization-induced lysosome membrane permeabilization (LMP), a novel mechanism involving the phosphorylation of MLKL, which leads to the formation of amyloid-like polymers. Crucial insights have been revealed through the research, including the occurrence of LMP before plasma membrane rupture and the participation of active lysosomal proteases. The colocalization of p-MLKL and LAMP2 underscores MLKL's role in inducing LMP. Highly purified lysosomes containing activated p-MLKL and MLKL polymers have been successfully isolated, highlighting their significance in the necroptotic process. Live-cell imaging showcased vesicle fusion before LMP. Encouragingly, the CTSB inhibitor CA-074Me exhibits potential for therapeutic use. These groundbreaking findings present exciting opportunities for targeted therapeutics and precision medicine approaches to effectively combat necroptosis-associated diseases.
Fig: (a)In live cell imaging, we observed HT-29 cells preloaded with Green Dextran beads and treated with T/S/Z, revealing that lysosome membrane permeabilization (LMP) occurs before plasma membrane rupture. b) The use of LysoTracker Red DND-99 and Sytox Green after T/S/Z treatment showed the release of active cathepsins into the cytosol, further confirming the occurrence of LMP. c) Through western blotting of cytosol and membrane fractions after DMSO or T/S/Z treatment, we identified MLKL and other indicators, providing evidence of MLKL's involvement in necroptosis. d) Activation of MLKL leads to its translocation to the lysosome membrane, initiating LMP and releasing active cathepsins into the cytosol, ultimately resulting in plasma membrane rupture.