Abstract
The canonical (caspase-1) and noncanonical (comprising caspases 4, 5 and 11; hereafter, caspase-4/5/11) inflammasomes both cleave gasdermin D (GSDMD) to induce pyroptosis1,2. Whereas caspase-1 processes IL-1β and IL-18 for maturation3,4,5,6, no cytokine target has been firmly established for lipopolysaccharide-activated caspase-4/5/117,8,9. Here we show that activated human caspase-4, but not mouse caspase-11, directly and efficiently processes IL-18 in vitro and during bacterial infections. Caspase-4 cleaves the same tetrapeptide site in pro-IL-18 as caspase-1. The crystal structure of the caspase-4–pro-IL-18 complex reveals a two-site (binary) substrate-recognition mechanism; the catalytic pocket engages the tetrapeptide, and a unique exosite that critically recognizes GSDMD10 similarly binds to a specific structure formed jointly by the propeptide and post-cleavage-site sequences in pro-IL-18. This binary recognition is also used by caspase-5 as well as caspase-1 to process pro-IL-18. In caspase-11, a structural deviation around the exosite underlies its inability to target pro-IL-18, which is restored by rationally designed mutations. The structure of pro-IL-18 features autoinhibitory interactions between the propeptide and the post-cleavage-site region, preventing recognition by the IL-18Rα receptor. Cleavage by caspase-1, -4 or -5 induces substantial conformational changes of IL-18 to generate two critical receptor-binding sites. Our study establishes IL-18 as a target of lipopolysaccharide-activated caspase-4/5. The finding is paradigm shifting in the understanding of noncanonical-inflammasome-mediated defences and also the function of IL-18 in immunity and disease.
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Acknowledgements
We thank Y. Chen for assistance with ITC experiments; and the staff of the BL02U1 beamline at the Shanghai Synchrotron Radiation Facility for assistance during data collection. This work was supported by the CAS Strategic Priority Research Program (XDB37030202 and XDB29020202), the Basic Science Center Project (82388201) of NSFC, the National Key R&D Program of China (2022YFA1304700), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019-I2M-5-084) and a grant from the Youth Innovation Promotion Association CAS. F.S. is also supported by the Tencent New Cornerstone Investigator Program.
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F.S. and J.D. conceived and supervised the study. X.S. made critical initial observations and performed all of the cellular experiments. Q.S., assisted by Y.H., determined the crystal structure and performed all of the in vitro assays. H.Z. provided technical assistance. Y.C., supervised by M.D., performed MS experiments. F.S. and J.D. analysed the data and wrote the manuscript. All of the authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Profiling inflammasome component expression in the NCI-60 and analyses of IL-18 maturation/secretion in caspase-4-activated cells.
a, Lysates of 56 cancer cell lines from the NCI-60 were analysed by immunoblotting using indicated antibodies. b, Selected IL-18-positive cells were stimulated with LPS electroporation with or without addition of zVAD. c, Immunoblotting profiling of NLRP3, ASC, caspase-1, caspase-4, and GSDMD expression in THP1, HEK 293 T, A431 and SiHa cells. d, WT, CASP4−/− (KO), or GSDMD−/− (KO) A431 cells were infected with S. Typhimurium (S.T.). e, f, WT or CASP4−/− (KO), or GSDMD−/− (KO) SiHa cells were infected with S. flexneri ΔospC3 (e) or S. Typhimurium (f). b, e, LDH release-based cell death measurement and ELISA-based IL-18 detection in culture supernatants are shown as means (bars) of three replicates (circles). d–f, Culture supernatants (Sup.) and cell lysates were separately analysed by immunoblotting. All data are representative of three independent experiments.
Extended Data Fig. 2 IL-18 processing induced by LPS-activated caspase-4 is defective in mice but effective in higher mammals.
a, Caspase-4 or caspase-11 was stably expressed in CASP4−/− (KO) HeLa cells. LDH release-based cell death data are shown as means (bars) of three replicates (circles). b, Caspase-11 was co-expressed with Flag-tagged human or mouse pro-IL-18 or mouse GSDMD in 293 T cells. a, b, Cells were stimulated with LPS electroporation. Cell supernatants (Sup.) (a) and lysates (a, b) were analysed by immunoblotting. c, d, LPS-primed primary (c) or immortalized (d) Gsdmd−/− (KO) BMDMs stably expressing mouse pro-IL-18-Flag (d) were stimulated with LPS electroporation to activate caspase-11 or LFn-BsaK plus protective antigen to activate the NAIP2-NLRC4 inflammasome. Cell lysates were analysed by immunoblotting. e, Pairs of caspase-4 and pro-IL-18 from human or other indicated mammals were co-expressed in 293 T cells. f, An indicated IL-1-family pro-cytokine with a C-terminal Flag tag was expressed in GSDMD−/− HeLa cells. e, f, Cells were stimulated with LPS electroporation, and cell lysates were analysed by immunoblotting. All data are representative of three independent experiments.
Extended Data Fig. 3 Comparison of caspase-4, −1, and −11 activity in cleaving pro-IL-18 and effect of OspC3ARD on caspase-4 processing of pro-IL-18.
a, Human or mouse pro-IL-18 was incubated with equivalent amounts of caspase-4/1-p20/p10 or caspase-11-p22/p10 for indicated times. b, Mouse pro-IL-18 was incubated with titrating relative molar amounts of caspase-11-p22/p10. Enz./Sub., enzyme/substrate. c, ITC measurements of the binding between caspase-4/11 and pro-IL-18. d, Human pro-IL-18 was incubated with caspase-4-p20/p10 alone or in the presence of excessive OspC3ARD (40 times of caspase-4). a, b, d, Reaction mixtures were subjected to SDS-PAGE analyses. e, Solution or crystal samples of the OspC3ARD–caspase-4-p20/p10-C/A–pro-IL-18 ternary complex were subjected to SDS-PAGE analyses. Data (a–d) are representative of three independent experiments.
Extended Data Fig. 4 Structural analyses of caspase-4–pro-IL-18 complex.
a, Structural comparison of the caspase-4–pro-IL-18 complex with the caspase-4–GSDMD-C binary complex (PDB code: 6KMZ). b, Superimposition of OspC3ARD–caspase-4-p20/p10 structure in the ternary complex with the reported OspC3ARD–caspase-4-p30 structure (PDB code: 7WR1). c, Superimposition of caspase-4-p20/p10 structure in the ternary complex with caspase-4-p20/p10 in the GSDMD-C complex (PDB code: 6KMZ). d, Sigma-A weighted 2Fo − Fc electron density map (contoured at 1.0 σ) of caspase-4-bound pro-IL-18 overlapped with the cartoon-loop model. e, Cartoon model of pro-IL-18 in the ternary complex. The caspase-4 cleavage-site D36 is shown as a magenta sphere, and the propeptide is in lemon. Residues involved in the interaction between the propeptide and the post-cleavage-site region are labelled and shown as sticks. f, Purified pro-IL-18 protein was treated with a Cys-Lys-specific crosslinker sulfo-GMBS (7.3 Å) under an optimized reaction condition and the samples were analysed by mass spectrometry. The table lists the identified crosslinked Cys-Lys pairs and the corresponding residues are mapped onto the pro-IL-18 structure on the left (cartoon model). C74 is located in a long flexible loop region, and thus showed highly efficient crosslinking with multiple distant residues. K103 and C112 are also located in relatively flexible regions, and a weak K103-C112 crosslinking signal appeared. The pro-IL-18 structure predicts two specific crosslinking pairs (C10-K148 and C10-K44), the latter of which did not appear due to a strong hydrogen bond between K44 and D8. g, Close-up view of the N terminus of caspase-4 p10 around the pro-IL-18-binding exosite interface. The pro-IL-18 structure is shown in surface scheme. The deduced extension from the N terminus of caspase-4 p10 is shown as dotted arrow and has clashes with the C-terminus of pro-IL-18.
Extended Data Fig. 5 Analyses of the binding interfaces between caspase-4 and pro-IL-18.
a, ITC profiles of the binding between caspase-4 (WT or an indicated mutant) and pro-IL-18 (WT or an indicated mutant). The C258A mutant was used for all caspase-4-p20/p10 in the assay. b, Calculated dissociation constant (KD) and binding stoichiometry (N) of ITC assays (a) are expressed as means ± s.d. from three determinations. ND, not detectable. c, e, Purified pro-IL-18 (WT or an indicated mutant) was incubated with caspase-4-p20/p10 (WT or an indicated mutant). Reaction mixtures were subjected to SDS-PAGE analyses. d, Approximate catalytic efficiency (kcat/Km) values of caspase-4 (WT or an indicate mutant), caspase-1, caspase-11, and caspase-5 in cleaving pro-IL-18. Purified pro-IL-18 was incubated with a titration series of the p20/p10 form of indicated caspases for 30 min. Reaction mixtures were subjected to SDS-PAGE analyses and quantification of the gel bands was performed to derive the kcat/Km. Calculated kcat/Km are expressed as means ± s.d. from three determinations. Mouse pro-IL-18 was assayed for caspase-11. f, Caspase-4 (WT or an indicated mutant) was co-expressed with pro-IL-18-Flag in CASP4−/−/GSDMD−/− (DKO) HeLa cells as in Fig. 3f. Cells were stimulated with LPS electroporation for 80 or 120 min, and cell lysates were analysed by immunoblotting. All data are representative of three independent experiments.
Extended Data Fig. 6 Analyses of the interaction between caspase-4 or caspase-3-cleaved IL-18 and IL-18Rα.
Gel-filtration chromatography analyses of complex formation between the extracellular domain of IL-18Rα and pro-IL-18, mature IL-18, caspase-4-cleaved pro-IL-18 (WT, Δ70-77, or F38D), or caspase-3-cleaved pro-IL-18. SDS-PAGE of the elution fractions are shown when formation of a complex was observed. All data are representative of three independent experiments.
Extended Data Fig. 7 Sequence and structural analyses of pro-IL-18 recognition by inflammatory caspases.
a, Sequence alignment of the protease-domains of inflammatory caspases. Identical residues are in red background and conserved ones are in red. Numbers of starting residues are indicated on the left. Residues responsible for caspase-4 binding to pro-IL-18 are conserved in caspase-5 and -1 but differ in caspase-11 as colour-highlighted. b, A modelled structure of the caspase-1–pro-IL-18 complex. The structure of caspase-1-p20/p10 in the GSDMD-C complex (PDB code: 6KN0) was overlaid with that of caspase-4-p20/p10 in the pro-IL-18 complex. Close-up view on the right shows binding interfaces mediated by the exosites in caspase-4 and caspase-1. Residues that bind pro-IL-18 are shown as sticks.
Supplementary information
Supplementary Information
Uncropped immunoblots for key data presented in Figs. 1–5 and Extended Data Figs. 1, 2 and 5.
Supplementary Video 1
Structural recognition of pro-IL-18 by caspase-4.
Supplementary Video 2
Conformational changes of the caspase-4-cleaved IL-18 to render an IL-18Rα-receptor-binding-competent state.
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Shi, X., Sun, Q., Hou, Y. et al. Recognition and maturation of IL-18 by caspase-4 noncanonical inflammasome. Nature 624, 442–450 (2023). https://doi.org/10.1038/s41586-023-06742-w
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DOI: https://doi.org/10.1038/s41586-023-06742-w
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