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Structural insights into cytokine cleavage by inflammatory caspase-4

A Publisher Correction to this article was published on 03 January 2024

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Abstract

Inflammatory caspases are key enzymes in mammalian innate immunity that control the processing and release of interleukin-1 (IL-1)-family cytokines1,2. Despite the biological importance, the structural basis for inflammatory caspase-mediated cytokine processing has remained unclear. To date, catalytic cleavage of IL-1-family members, including pro-IL-1β and pro-IL-18, has been attributed primarily to caspase-1 activities within canonical inflammasomes3. Here we demonstrate that the lipopolysaccharide receptor caspase-4 from humans and other mammalian species (except rodents) can cleave pro-IL-18 with an efficiency similar to pro-IL-1β and pro-IL-18 cleavage by the prototypical IL-1-converting enzyme caspase-1. This ability of caspase-4 to cleave pro-IL-18, combined with its previously defined ability to cleave and activate the lytic pore-forming protein gasdermin D (GSDMD)4,5, enables human cells to bypass the need for canonical inflammasomes and caspase-1 for IL-18 release. The structure of the caspase-4–pro-IL-18 complex determined using cryogenic electron microscopy reveals that pro-lL-18 interacts with caspase-4 through two distinct interfaces: a protease exosite and an interface at the caspase-4 active site involving residues in the pro-domain of pro-IL-18, including the tetrapeptide caspase-recognition sequence6. The mechanisms revealed for cytokine substrate capture and cleavage differ from those observed for the caspase substrate GSDMD7,8. These findings provide a structural framework for the discussion of caspase activities in health and disease.

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Fig. 1: Human caspase-4 can efficiently cleave human pro-IL-18 but not other IL-1-family cytokines.
Fig. 2: Overview of the caspase-4–pro-IL-18 complex.
Fig. 3: Cryo-EM map and model of the caspase-4–pro-IL-18 complex.
Fig. 4: Electrostatic interactions between the pro-IL-18 pro-domain and caspase-4 promote cytokine processing.
Fig. 5: A hydrophobic exosite is required for recognition and cleavage of pro-IL-18 by caspase-4.
Fig. 6: Engineering of caspase-11 into an efficient IL-18-converting enzyme.

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Data availability

All data and materials reported in the Article and its Supplementary Information are available on request. Raw data needed to recreate plots and uncropped gel images are accessible in the Supplementary Information. The electron density maps of caspase-4–pro-IL-18 at 3.2 Å have been deposited at the Electron Microscopy Data Bank (EMDB) under accession code EMD-40678. The atomic coordinates for caspase-4–pro-IL-18 have been deposited at the PDB under accession code 8SPB. Atomic coordinates for mature IL-18 and caspase-11 were downloaded from the PDB under accession codes 3WO2 and 6KN1, respectively. Source data are provided with this paper.

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Acknowledgements

We thank D. Okin for providing primary cells and other members of the Kagan and Wu laboratories for discussions; T. Sam Xiao for the caspase-1 expression plasmid and I. Brodsky for the Salmonella strain; R. Walsh, S. Sterling. M. Mayer and S. Rawson for cryo-EM grid screening and data collection, and K. Song for cryo-EM grid screening and dataset collection; and the staff at SBGrid for software and computing support. This work was supported by NIH grants AI67993, AI116550 and P30DK34854 (to J.C.K.), AI124491 and AI139914 (to H.W.), a PhD fellowship by the Boehringer Ingelheim Fonds (to P.D.) and a postdoctoral fellowship from the Charles A. King Trust (to Y.D.). We acknowledge the International Union of Crystallography on the occasion of its 75th anniversary.

Author information

Authors and Affiliations

Authors

Contributions

P.D., Y.D., J.C.K. and H.W. conceived the study and designed experiments. P.D. and W.M. designed and cloned constructs. P.D. and W.M. performed preliminary expression and purification studies. P.D. purified proteins and performed biochemical assays. P.D. performed cell-based experiments. P.D. purified the caspase-4–pro-IL-18 complex for structural studies. Y.D. and P.D. generated cryo-EM grids for data collection. Y.D. screened cryo-EM grids and collected cryo-EM data. Y.D. analysed cryo-EM data and performed model building and refinement. J.M. performed cross-linking MS and data analysis under the supervision of S.P.G. P.D., Y.D., J.C.K. and H.W. wrote the manuscript with input from all of the other authors.

Corresponding authors

Correspondence to Hao Wu or Jonathan C. Kagan.

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Competing interests

J.C.K. consults and holds equity in Corner Therapeutics, Larkspur Biosciences and Neumora Therapeutics. H.W. is a co-founder and chair of the scientific advisory board of Ventus Therapeutics. None of these relationships influenced this study. The other authors declare no competing interests.

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Nature thanks Osamu Nureki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Comparative analysis of IL-1 cytokine cleavage across mammals.

a, 10 µg of indicated proteins were separated by SDS-PAGE and stained with InstantBlue. b-k, In vitro cleavage of pro-forms of indicated human IL-1 family cytokines by caspase-4, caspase-1 and caspase-5. Immunoblots are representative of three independent repeats. Asterisk marks signal due to cross-reaction of primary antibody with His-tagged p20 subunit of the caspase. l,m, Immunoblots showing in vitro cleavage of human pro-IL-1β by human caspase-1 and caspase-5. Asterisk marks signal due to cross-reaction of primary antibody with His-tagged p20 subunit of the caspase. n-q, Immunoblots showing in vitro cleavage of murine pro-IL-18 by human caspase-1 and caspase-4, and murine caspase-1 and caspase-11. rv, Immunoblots showing in vitro cleavage of pro-IL-18 from indicated mammalian species by caspase-4 homologue from the same species. w, x, Immunoblot and quantification of in vitro cleavage of human pro-IL-18 mutants by murine caspase-11. n = 4 biological replicates for caspase-4 and n = 3 biological replicates for caspase-11. All immunoblots are representative of at least three biological replicates. Bars and error bars represent mean ± SEM. Statistical significance was determined by unpaired, two-sided student’s t-test. For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 2 Cytosolic LPS induces NLRP3-independent IL-18 release from human cells.

a, b, WT or NLRP3-deficient THP1 monocytes were primed with Pam3CSK4, or left unprimed, and electroporated with LPS (or PBS) in presence or absence of MCC950 and LDH and IL-18 release into supernatant was quantified after 2 h. c, ELISA analysis of purified pro-IL-18 and mature IL-18. Mature IL-18 was generated by cleavage of pro-IL-18 with recombinant caspase and complete cleavage was confirmed by immunoblot. Immunoblot and ELISA results are representative of two experiments. d, e, WT or NLRP3-deficient THP1 monocytes were electroporated with LPS (or PBS) in the presence of absence of MCC950. IL-18 from cell culture supernatants was immunoprecipitated and analysed by immunoblot. f, g, Pam3CSK4-primed WT or NLRP3-deficient THP1 monocytes were electroporated with LPS (or PBS) in the presence of absence of MCC950. IL-1β was immunoprecipitated from supernatants and analysed by immunoblot. h, Immunoblot analysis of THP1 cells in which expression of caspase-1 or caspase-4 was disrupted by CRISPR–Cas9. i, j, LPS-primed THP1 macrophages deficient for caspase-1, caspase-4 or treated with a non-target sgRNA (NT) were electroporated with LPS (or PBS) and LDH release and IL-18 levels in supernatants were quantified after 2 h. IL-1β from supernatants was immunoprecipitated and analysed by immunoblot. Immunoblot is representative of three biological replicates. k, Immunoblot analysis of LPS-primed caspase-1 or caspase-4-deficient THP1 macrophages compared to caspase-4-deficient THP1 cells reconstituted with caspase-5 by retroviral transduction. Cells were differentiated into macrophages and stimulated with LPS for 4 h. l, Immunoblot analysis of caspase-4-deficient THP1 cells reconstituted with caspase-4 or caspase-5 by retroviral transduction. m, Caspase-4-deficient THP1 macrophages expressing GFP only, caspase-4 or caspase-5 were primed with LPS before delivery of LPS into the cytosol by electroporation. LDH and IL-18 release into supernatants was quantified after 2 h. n, WT or caspase-4-deficient THP1 macrophages were infected with a flagellin-deficient strain of Salmonella and LDH and IL-18 release was quantified after 24 h. Immunoblots are representative of three (d, e, f, g, h, j) or two (h, l, k, c) biological replicates. Bars and error bars represent mean ± SEM of three (i, n), four (a, b right panels) or five (a, b left panel) biological replicates. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test: ns = not significant (p > 0.05). For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 3 Cytosolic LPS-induced IL-18 release from murine cells is NLRP3-dependent.

a, b, c, WT or NLRP3-deficient iBMDMs were primed with extracellular LPS, or left unprimed, and electroporated with LPS (or PBS) in the presence of absence of MCC950. LDH, IL-18 and IL-1β release into supernatant was quantified after 2 h. n = 3 biological replicates. d, e, LPS-primed WT iBMDMs were electroporated with LPS (or PBS) in the presence or absence of MCC950. IL-18 and IL-1β were immunoprecipitated from supernatants and analysed by immunoblot (representative of three biological replicates). f, Immunoblot analysis caspase-4-deficient THP1 cells reconstituted with caspase-11 by retroviral transduction (representative of two independent repeats). g, Caspase-4-deficient THP1 macrophages expressing caspase-11 were primed with extracellular LPS, or left unprimed, and electroporated with LPS (or PBS) in the presence of absence of MCC950. LDH, and IL-18 release into supernatant was quantified. n = 6 biological replicates. Bars and error bars represent mean ± SEM. Statistical significance was determined by two-way ANOVA (a,b,c) or one-way ANOVA (g) with Tukey’s multiple comparisons test: ns = not significant (p > 0.05). For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 4 Assembly and purification of a recombinant caspase-4/pro-IL-18 complex.

a, SEC profile and corresponding coomassie-stained SDS-PAGE gel of purified pro-IL-18 expressed in insect cells. b, SEC profile and corresponding coomassie-stained SDS-PAGE gel of purified catalytically inactive caspase-4 p20/p10 expressed in E. coli. c, SEC profile and corresponding coomassie-stained SDS-PAGE gel of a complex consisting of caspase-4 p20/p10 and pro-IL-18. Complex was assembled by co-incubation at 37 °C for 20 min. d, SEC profile and corresponding coomassie-stained Blue Native-PAGE gel of a complex consisting of caspase-4 p20/p10 and pro-IL-18 crosslinked with BS3. Gels and SEC profiles are representative of at least two independent purifications. Peak fractions that were combined for downstream applications are highlighted in blue. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5 Cryo-EM data processing for the caspase-4/pro-IL-18 complex.

a, Cryo-EM raw image (representative of two data collections). b, Cryo-EM data processing flow chart. The black arrow pointing to “State C” shows domain flexibility indicated by 3D variability analysis. c, Heat map for the orientation of particles used for the final reconstruction. d, Fourier shell correlation (FSC) plots. e, 3D FSC plot.

Extended Data Fig. 6 Crosslinking mass spectrometry analysis of caspase-4/pro-IL-18 complex and structural comparison between caspase-4-bound pro-IL-18 and free IL-18.

a, Summary of BS3 crosslinking between caspase-4 and pro-IL-18. The crosslinked peptides with high confidence are shown with residue ranges, and colour labelled by their domain colour except for the crosslinked Lys residues which are in red. b, Crosslinked lysine pairs between caspase-4 and pro-IL-18 mapped onto the structure of the caspase-4/pro-IL-18 complex and indicated by black dash lines. c, The molecular interaction of the active form of caspase-4 interacting with pro-IL-18. Model was derived by replacing Ala258 with Cys. d, e, Structural alignment between pro-IL-18 bound to caspase-4 and the crystal structure of mature IL-18 (d) and in topology diagrams (e). β-strands are labelled sequentially with those in the pro-domain denoted by a prime (‘). f, Crosslinked lysine pairs within pro-IL-18 in the caspase-4/pro-IL-18 complex (upper table), and those within pro-IL-18 alone before incubating with caspase-4 (lower table). The former pairs are consistent with the caspase-4/pro-IL-18 complex structure, but not the mature IL-18 structure, and the latter pairs are not consistent with any IL-18 structures, suggesting that the pro-IL-18 conformation may be different before caspase-4 binding. g, Crosslinked lysine pairs within pro-IL-18 mapped onto the structure of the caspase-4/pro-IL-18 complex and indicated by black dash lines. h, AlphaFold predicted model of pro-IL-18 coloured by per-residue pLDDT score, ranging from unreliable in red to reliable in blue. Residues 16-25 of the pro-domain form a β-hairpin structure, consistent with our cryo-EM structure pro-IL-18 in complex with caspase-4.

Source Data

Extended Data Fig. 7 The pro-domain of pro-IL-18 mediates the interaction with caspase-4.

a, c, Immunoblots showing in vitro cleavage of pro-IL-18 mutants by caspase-4. The pro-domain chimera consists of the pro-domain of murine pro-IL-18 fused to the mature domain of human pro-IL-18. Immunoblots are representative of three biological replicates. b, Sequence alignment of human and murine pro-IL-18. Identical and similar amino acids are highlighted in red or white boxes, respectively. Sequences were aligned using ClustalOmega online tool and plotted in ESPript 3.0. d-h, Immunoblots showing in vitro cleavage of murine pro-IL-18 mutants by caspase-4. i, ITC analysis of binding of mature IL-18 to WT caspase-4. j, Immunoblots showing in vitro cleavage of human pro-IL-18 ΔN24 by caspase-4. Pro-IL-18 ΔN24 lacks the first 24 amino acids at the N-terminus. k, Analytical SEC demonstrating no binding between pro-IL-18 ΔN24 and caspase-4. All data are representative of three independent repeats. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 8 Biochemical characterization of caspase-4 and pro-IL-18 mutants that disrupt interaction interfaces with pro-IL-18.

a, ITC analysis of binding of pro-IL-18 (purified from insect cells) to caspase-4 mutants. b, Thermodynamic parameters of binding of indicated caspase-4 mutants to pro-IL-18 as determined by ITC. c, d, e, Immunoblots showing in vitro cleavage of human pro-IL-18 by indicated caspase-4 mutants. f, ITC analysis of binding of indicated pro-IL-18 mutants (purified from E. coli) to WT caspase-4. Graphs are representative of at least three biological replicates. g, Thermodynamic parameters of binding of caspase-4 mutants to pro-IL-18 as determined by ITC. h-k, Immunoblots showing in vitro cleavage of indicated human pro-IL-18 variants by caspase-4. Immunoblots are representative of three biological replicates. ITC graphs are representative of three independent repeats. ITC results represent mean ± SD of three independent measurements. n.d. = no binding detected. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 9 Effect of structure-based mutations in caspase-4 on GSDMD and pro-IL-18 cleavage in cells.

a, Expression of indicated caspase-4 mutants in caspase-4-deficient THP1 cells (representative of two independent repeats). b, In vitro cleavage of human GSDMD by caspase-4 mutants. cf, Immunoblots showing in vitro cleavage of pro-IL-18 by caspase-4 mutants. Immunoblots are representative of three independent repeats. g, h, Caspase-4-deficient THP1 macrophages expressing caspase-4 mutants were primed with LPS and electroporated with LPS (or PBS). Processing of GSDMD and pro-IL-18 was analysed by immunoblot. Immunoblots are representative of three biological replicates. i, Immunoblot showing expression of indicated pro-IL-18 mutants in IL-18-deficient THP1 cells (representative of two independent repeats). j, IL-18-deficient THP1 cells expressing pro-IL-18 mutants were electroporated with LPS (or PBS) in the presence of MCC950 and release of LDH into cell culture supernatant was quantified after 2 h. k, ELISA analysis demonstrating that pro-IL-18V47N/I48N is not recognized by the used ELISA reagent. ELISA results are displayed as mean ± SD of two technical replicates and are representative of two independent biological replicates. Bars and error bars in b, j represent mean ± SEM of three biological replicates. Each data point represents result of one independent experiment. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test: ns = not significant (p > 0.05). For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 10 Mutagenesis of murine caspase-11 based on structural and sequence alignments.

a, Sequence alignment of human caspase-4 and murine caspase-11. Identical and similar amino acids are highlighted in red or white boxes, respectively. Sequences were aligned using ClustalOmega and plotted in ESPript 3.0. b, c, d, Immunoblots showing in vitro cleavage of human pro-IL-18 by caspase-11 mutants. Immunoblots are representative of three biological replicates. For gel source data, see Supplementary Fig. 1.

Extended Data Table 1 Data collection, data processing and validation statistics

Supplementary information

Supplementary Figure 1

Uncropped gels and blots.

Reporting Summary

Supplementary Video 1

3D variability analysis of caspase-4–pro-IL-18 complex.

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Devant, P., Dong, Y., Mintseris, J. et al. Structural insights into cytokine cleavage by inflammatory caspase-4. Nature 624, 451–459 (2023). https://doi.org/10.1038/s41586-023-06751-9

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