Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1

J Biol Chem. 2018 May 4;293(18):7058-7067. doi: 10.1074/jbc.RA117.001329. Epub 2018 Feb 6.


Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined.

Keywords: GSDMD; caspase; cell death; enzyme kinetics; fluorescence; interleukin; proptosis.

Publication types

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

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / metabolism
  • Caspase 1 / metabolism*
  • Caspases / metabolism*
  • Caspases, Initiator
  • Cell Death
  • Cytokines / metabolism
  • Inflammation Mediators / metabolism
  • Interleukin-10 / metabolism
  • Interleukin-1beta / metabolism
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Peptides / metabolism*
  • Phosphate-Binding Proteins
  • Proteolysis
  • Recombinant Proteins / metabolism
  • Substrate Specificity


  • Apoptosis Regulatory Proteins
  • Cytokines
  • Gsdmd protein, mouse
  • Inflammation Mediators
  • Interleukin-1beta
  • Intracellular Signaling Peptides and Proteins
  • Peptides
  • Phosphate-Binding Proteins
  • Recombinant Proteins
  • Interleukin-10
  • Casp4 protein, mouse
  • Caspases
  • Caspases, Initiator
  • Casp1 protein, mouse
  • Caspase 1