Inflammatory caspase substrate specificities

Abstract

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.

Keywords: IL-18; IL-1β; caspase substrates; caspase-1; caspase-11; caspase-4; caspase-5; cytokines; inflammasomes; inflammatory caspases; pyroptosis.

Fig 1

Overview of canonical and non-canonical inflammasome activation pathways. (A) Diverse stimuli are sensed by pattern-recognition receptors (PRRs) which recruit ASC and pro-caspase-1 (pro-CASP1) to form the canonical CASP1 inflammasome. CASP1 undergoes autoproteolytic maturation then cleaves and activates gasdermin D (GSDMD) and the interleukin family of cytokines IL-1β and IL-18. CASP1-mediated cleavage of IL-1β and IL-18 generates 17 and 18 kDa species, respectively, that are biologically active and signal through the IL-1 and IL18 receptors respectively. (B) Pro-caspases-4/5 (Pro-CASP4/5) bind to lipopolysaccharide (LPS) directly, leading to formation of the CASP4/5 non-canonical inflammasome. CASP4/5 undergo autoproteolytic maturation to cleave and activate GSDMD and IL-18 but cleave IL-1β into a biologically inactive 27 kDa species that does not signal to the IL-1 receptor. (C) Pro-caspase-11 (Pro-CASP11) binds directly to LPS and is first activated by autoproteolysis, and subsequently forms the non-canonical CASP11 inflammasome. Active CASP11 cleaves and activates GSDMD but cleaves and inactivates IL-1β by generating IL-1β p27. Image created using Biorender.

Fig 2

Schematic of inflammatory caspase peptide and protein substrate specificities. (A) The residue cleaved by caspases (dotted line) is designated P₁ and the residues N-terminal to the cleaved peptide bond are numbered consecutively from the P₁ position. Residues to the C-terminus of the peptide bond are also number consecutively and designate as prime (e.g., P₁′) residues. These residues interact with the caspase substrate binding pockets, which have corresponding S designations (e.g., S₄ – S₁) to match the substrate convention, that help fine tune specificity. The preferred peptide substrate sequence and position of residues for the inflammatory caspases, as determined by positional scanning peptide library studies, are depicted. (B) Protein substrates indicating the amino acid sequence and position of caspase cleavage is shown. The biological consequence of cleavage at each specific site is also indicated. (C) Putative substrates that have potential crosstalk with other cell death pathways and the caspase reported to cleave those substrates are listed. These substrates need to be verified for direct processing in biologically relevant contexts. Image created using Biorender.