CASP5-mediated substrate cleavage

CASP5-mediated substrate cleavage Caspase-5 (CASP5), like CASP4, is activated by cytosolic bacterial lipopolysaccharide (LPS). Once activated, CASP5 cleaves gasdermin D (GSDMD), a substrate also targeted by CASP1, CASP4, and Casp11, a murine homolog of human CASP4 and CASP5 (Shi J et al., 2014, 2015; Kayagaki N et al., 2015; Wang K et al., 2020). The cleavage at aspartic acid residue 275 (D275) within the central linker region of GSDMD releases a cytotoxic 31-kDa N-terminal fragment (GSDMD(1–275)) and a 22-kDa C-terminal fragment (GSDMD(276–484)). The N-terminal fragment has pore-forming activity, inserting into lipid membranes to form 10–16 nm pores, which ultimately drive pyroptotic cell death (Liu X et al., 2016; Ding J et al., 2016; Sborgi L et al., 2016; Aglietti RA et al., 2016; Feng S et al., 2018). The C-terminal fragment maintains an autoinhibitory interaction with the N-terminal domain in full-length GSDMD, and its cleavage by inflammatory caspases, CASP1, CASP4, or CASP5, releases this inhibition, enabling pyroptosis (Shi J et al. 2015; Ding J et al. 2016; Liu Z et al. 2019; Yang J et al. 2018; Kuang S et al. 2017; Wang K et al., 2020). Similar to CASP4, CASP5 directly cleaves pro-inflammatory cytokines of the interleukin-1 (IL-1) family, including pro-IL-18 and pro-IL-1β, exhibiting distinct substrate preferences (reviewed by Exconde PM, 2024). CASP5 efficiently cleaves pro-IL-18 at D36, producing the mature, biologically active cytokine (Shi X et al., 2023; Devant P et al., 2023; Exconde PM et al., 2023). Structural and biochemical analyses revealed that this cleavage relies on a bivalent recognition mechanism, in which pro-IL-18 binds CASP4/CASP5 through two interfaces: the protease exosite binds a hydrophobic pocket within pro-IL-18, while the active site of caspase engages charged residues located within and adjacent to the tetrapeptide recognition motif in the pro-domain (Shi X et al., 2023; Devant P et al., 2023). In contrast, CASP5 cleaves pro–IL‑1β at D27, producing an inactive fragment that lacks receptor-stimulating activity (Exconde PM et al., 2023; reviewed by Exconde PM, 2024). CASP5 may also contribute to pro-IL-1α processing and maturation (Wiggins KA et al., 2019). Mature IL-1 cytokines are released through GSDMD pores, amplifying the inflammatory response in mammals (Shi J et al., 2015; Kayagaki N et al., 2015; reviewed by Broz P et al., 2020; Liu X et al., 2021).