Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11

J Biol Chem. 2020 Aug 7;295(32):11292-11302. doi: 10.1074/jbc.RA120.014259. Epub 2020 Jun 18.


Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.

Keywords: caspase cytokine; caspase-1; caspase-11; cell death; cysteine protease; gasdermin D; inflammation; inflammatory caspase; innate immunity; interleukin-1 (IL1); pyroptosis; substrate specificity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caspases / metabolism*
  • Catalysis
  • Humans
  • Immunity, Innate
  • Inflammation / metabolism*
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Intracellular Signaling Peptides and Proteins / physiology*
  • Mice
  • Phosphate-Binding Proteins / chemistry
  • Phosphate-Binding Proteins / metabolism
  • Phosphate-Binding Proteins / physiology*
  • Proteolysis
  • Pyroptosis*
  • Substrate Specificity


  • GSDMD protein, human
  • Intracellular Signaling Peptides and Proteins
  • Phosphate-Binding Proteins
  • Caspases
  • caspase 11, human