Distinct necrotic protein cleavages define terminal events in necrosis

Apoptosis is executed by caspase-mediated proteolytic cleavage. In contrast, we know very little regarding the involvement of proteolytic cleavage in necrosis. Here, we systematically characterized the morphological and biochemical features of terminal events in necrotic cell death, defined by irreversible plasma membrane rupture, independent of upstream death-initiating pathways. Using three-dimensional optical diffraction tomography, we identified distinct nuclear morphological changes that distinguish necrosis from apoptosis and reveal heterogeneity in pyroptotic execution, with individual cells exhibiting either apoptotic or necrotic morphology. Consistent with these differences, we uncovered the necrosis-specific lamin-B1 cleavage pattern distinct from apoptotic processing. Using neo-N-terminomic analysis, we identified a substantial set of protein substrates proteolytically cleaved after Arg/Lys residues in necrosis mediated by extracellular trypsin-like proteases. These cleavage events drive nuclear structural remodeling, chromatin DNA degradation, and efficient phagocytic clearance of necrotic cell remnants, thereby limiting autoimmune pathology. Finally, we developed monoclonal antibodies recognizing specific necrotic cleavage events as biomarkers for the terminal events of necrosis in cells and in vivo.