title: GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes.
abstract: Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca(2+) from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, CA 94080;
authors.forename: Robin A
authors.initials: RA
authors.lastname: Aglietti
authors.affiliation: Department of Structural Biology, Genentech, Inc., South San Francisco, CA 94080;
authors.forename: Alberto
authors.initials: A
authors.lastname: Estevez
authors.affiliation: Department of Physiological Chemistry, Genentech, Inc., South San Francisco, CA 94080;
authors.forename: Aaron
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, CA 92037;
authors.forename: Monica Gonzalez
authors.initials: MG
authors.lastname: Ramirez
authors.affiliation: Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA 94080.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Liu
authors.affiliation: Department of Physiological Chemistry, Genentech, Inc., South San Francisco, CA 94080;
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Kayagaki
authors.affiliation: Department of Structural Biology, Genentech, Inc., South San Francisco, CA 94080;
authors.forename: Claudio
authors.initials: C
authors.lastname: Ciferri
authors.affiliation: Department of Physiological Chemistry, Genentech, Inc., South San Francisco, CA 94080; dixit.vishva@gene.com dueber.erin@gene.com.
authors.forename: Vishva M
authors.initials: VM
authors.lastname: Dixit
authors.affiliation: Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, CA 94080; dixit.vishva@gene.com dueber.erin@gene.com.
authors.forename: Erin C
authors.initials: EC
authors.lastname: Dueber
mesh_terms: D000818:Animals; D020169:Caspases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008081:Liposomes; D051379:Mice; D009154:Mutation; D009363:Neoplasm Proteins / Q000502:physiology*; D028044:Phosphate-Binding Proteins; D000069292:Pyroptosis; D000094524:Gasdermins
chemical_list: D020169:Caspases; D047908:Intracellular Signaling Peptides and Proteins; D008081:Liposomes; D009363:Neoplasm Proteins; D028044:Phosphate-Binding Proteins; C498366:GSDMD protein, human; D000094524:Gasdermins; C000624929:caspase 11, human
keywords: GsdmD; caspase-11; pyroptosis