Caspase-3 activation is a brake in GSDMD-mediated pyroptosis

Pyroptosis is a form of programmed cell death mediated by gasdermin proteins, with GSDMD and GSDME being the most extensively studied. Inflammatory caspases-1 or caspases-4/5/11 cleave GSDMD and release its pore-forming fragment GSDMD-NT, whereas the apoptotic caspase-3, cleaves GSDME and releases its pore-forming fragment GSDME-NT. In this study, we observed that caspase-3 is activated during GSDMD-mediated pyroptosis. Interestingly, downregulation of caspase-3 activity either through RNAi or caspase-3 inhibitor significantly increased cell death. Furthermore, we found that caspase-3 physically interacts with GSDMD-NT and cleaves it at a site distinct from those targeted by caspase-1 or caspases-4/5/11. This alternative cleavage generates a non-functional fragment of GSDMD-NT, thereby disrupting its integrity. Consequently, we demonstrate that caspase-3 activation serves as a negative feedback mechanism to regulate the intensity of GSDMD-NT-mediated pyroptosis.