Elucidation of the role of GSDME during apoptosis-driven secondary necrosis

Although clear evidence exists for the contribution of gasdermin E (GSDME) to hearing loss and tumor biology, the physiological function of GSDME and how it could explain a role in both disorders remained unclear. As GSDME was reported to be a substrate of the apoptotic caspase-3 and is proposed to act as a pore-forming molecule, we aimed in this thesis to unravel the role of GSDME in apoptosis-driven secondary necrosis. Therefore we explored different aspects both in silico and in vitro. Using homology-based modeling, we investigated the composition and functionality of secondary structures in the GSDME protein and compared our model to the published structures of other gasdermin (GSDM) proteins. Next, we measured the GSDME-dependent nuclear staining by DNA dyes and the influx of Texas Red-labeled dextrans during apoptosis-driven secondary necrosis in order to gain more insight about the kinetics and size characteristics of GSDME-dependent cell death. Finally, we monitored the cellular localization of GSDME during apoptosis-driven secondary necrosis using live-cell imaging. Our results show that GSDME facilitates the quick permeabilization of the plasma membrane after activation of caspase-3 favoring the staining by SYTOX dyes and Texas Red-labeled dextrans. However, we also found that GSDME is probably not the only executor of membrane permeabilization acting downstream of caspase-3 activation, as the nuclear staining by 7-AAD and efflux of FITC-labeled dextrans happened independent of GSDME expression during apoptosis-driven secondary necrosis. Future research should focus on the consequences of GSDME-mediated plasma membrane permeabilization in terms of release of pro-inflammatory molecules and clearance by phagocytic cells. In addition, our results question the universality of the barrel-stave pore model as the pore-forming mechanism of GSDM proteins. Further comparative analysis with other known pore-forming molecules should be done to provide more certainty about the pore-forming mechanism of GSDME.

Publication year:2021

Keywords:Doctoral thesis

Accessibility:Open