Activation of the inflammatory caspase‑4 (CASP4) is induced upon sensing of intracellular bacterial lipopolysaccharide (LPS) (Kajiwara Y et al. 2014; Casson CN et al. 2015; Vigano E et al. 2015; Lagrange B et al. 2018). Activated CASP4 drives non‑canonical inflammasome responses to fight bacterial infections (reviewed in Zamyatina A & Heine H 2020; Downs KP et al. 2020). LPS triggers auto‑processing at D289 in the inter‑subunit linker of CASP4, generating a CASP4 subunit p10 (290‑377) (Wang K et al. 2020). The CASP4 autoprocessing at D289 was required for induction of gasdermin D (GSDMD) cleavage thus promoting pyroptosis in human CASP4‑/‑ epidermoid carcinoma A431 and HeLa cells that stably expressed Flag‑tagged CASP4 (Wang K et al. 2020). Human CASP5 is thought to function similarly to CASP4 (Vigano E et al. 2015; Shi J et al. 2015). The protease activity of CASP5 can initiate pyroptosis trough processing of GSDMD at D275, which is covered by this Reactome annotation. However, the mechanisms underlying activation and LPS recognition by CASP5 require further study. Further, structural studies suggest that binding of GSDMD to CASP4 allosterically enhanced the catalytic activity of CASP4 by stabilizing the dimeric form of processed CASP4 (2xp10:p20) (Wang K et al. 2020). Once activated, CASP4, CASP5 cleave GSDMD at D275 within the central linker region generating a 31‑kDa N‑terminal fragment (GSDMD (1‑275)) which has an intrinsic pore‑forming activity to initiate pyroptosis and a 22‑kDa C‑terminal fragment (GSDMD (276‑484)) which inhibits cell death through intramolecular domain association (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). The expression of GSDMD (1‑275) in human embryonic kidney 293 (HEK293) cells induced pyroptosis, whereas overexpression of the C-terminal fragment of GSDMD (276-484) blocked cell death (Shi J et al. 2015). In addition, biochemical and structural studies of human GSDMD and mouse GSDMA3 showed the auto‑inhibitory conformation of gasdermin domains which is released upon interdomain cleavage by inflammatory caspases, including CASP4 (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). Thus, the CASP4, CASP5‑mediated cleavage is thought to release the cytotoxic GSDMD (1‑275) from intramolecular autoinhibition mediated by the C‑terminal fragment of GSDMD. The N‑terminal domain of GSDMD (1‑275) binds and inserts into lipid membranes where it assembles into pores 10‑16 nm in diameter (Ding J et al. 2016; Sborgi L et al. 2016). GSDMD pores facilitate the secretion of active forms of interleukin‑1β (IL‑1β) and IL‑18 from pyroptotic cells (Shi J et al. 2015; Ding J et al. 2016; Evavold CL et al. 2018). "The increasing abundance of membrane pores ultimately leads to membrane rupture and pyroptosis, releasing the entire cellular content" ‑ Feng S et al. 2018. The murine homolog of CASP4/5, CASP11, can also cleave GSDMD (Kayagaki N et al. 2015).
This Reactome event shows CASP4/CASP5‑mediated cleavage of GSDMD at D275.