title: GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death.
abstract: Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome-inserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Sborgi
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Rühl
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland.
authors.forename: Estefania
authors.identifier: 0000-0002-7074-2371
authors.initials: E
authors.lastname: Mulvihill
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Joka
authors.initials: J
authors.lastname: Pipercevic
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Rosalie
authors.initials: R
authors.lastname: Heilig
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Henning
authors.identifier: 0000-0002-1185-4592
authors.initials: H
authors.lastname: Stahlberg
authors.affiliation: Novartis Institutes for BioMedical Research Forum 1, Basel, Switzerland.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Farady
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Müller
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland petr.broz@unibas.ch sebastian.hiller@unibas.ch.
authors.forename: Petr
authors.initials: P
authors.lastname: Broz
authors.affiliation: Biozentrum, University of Basel, Basel, Switzerland petr.broz@unibas.ch sebastian.hiller@unibas.ch.
authors.forename: Sebastian
authors.identifier: 0000-0002-6709-4684
authors.initials: S
authors.lastname: Hiller
mesh_terms: D020170:Caspase 1 / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D020285:Cryoelectron Microscopy; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008081:Liposomes / Q000737:chemistry / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D009363:Neoplasm Proteins / Q000378:metabolism*; D010539:Permeability; D028044:Phosphate-Binding Proteins; D000069292:Pyroptosis; D000094524:Gasdermins
chemical_list: D020170:Caspase 1; D047908:Intracellular Signaling Peptides and Proteins; D008081:Liposomes; D009363:Neoplasm Proteins; D028044:Phosphate-Binding Proteins; C498366:GSDMD protein, human; D000094524:Gasdermins
keywords: atomic force microscopy; cell death; gasdermin; inflammasomes; inflammation; pyroptosis