Ubiquitination of gasdermin D N-terminal domain directs its membrane translocation and pore formation during pyroptosis

Gasdermin D (GSDMD) is a critical mediator of pyroptosis, which consists of a N-terminal pore-forming domain and a C-terminal autoinhibitory domain. The free N-terminal domain (GD-NT), which is released through caspase-1/11 cleavage, exhibits distinct features from the full-length GSDMD (GD-FL), including oligomerization, membrane translocation and pore-formation. However, the underlying mechanisms are not well clarified. Here, we found that GD-NT, but GD-FL, was massively ubiquitinated in cells. The K63-linked polyubiquitination of GD-NT at Lys236/237 (human/mouse), catalyzed by TRAF1, directly its membrane translocation and pore-formation during pyroptosis. Inhibition of GD-NT ubiquitination via site mutation or the UBA1 inhibitor PYR-41 suppressed cell death in several pyroptosis cell models. Additionally, the application of PYR-41 in septic mice efficiently suppressed the release of IL-18 and TNF-⍺. Thus, GD-NT ubiquitination is a key regulatory mechanism controlling its membrane localization and activation, which may provide a novel target for modulating immune activity in pyroptosis-related diseases.