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Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome

Nature volume 622, pages 188–194 (2023)Cite this article

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Abstract

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2,3,4,5,6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

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Fig. 1: TRX interacts with NLRP1.
Fig. 2: Architecture of the autoinhibited NLRP1–TRX complex.
Fig. 3: Molecular basis for TRX binding to NLRP1.
Fig. 4: TRX suppresses NLRP1 inflammasome activation.

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Data availability

All data needed to evaluate the conclusions in the paper are presented in the paper and/or extended data figures and table. Additional data and resources related to this paper may be requested from the authors. The cryo-EM maps and related structure coordinates of the NLRP1–sfTRX complex have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-32484 (consensus map) and EMD-35591 (focused map) and in the Protein Data Bank (PDB) under accession 7WGE. Source data are provided with this paper.

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Acknowledgements

The authors thank M. Kikkawa, H. Yanagisawa, A. Tsutsumi, Y. Sakamaki and Y. Kise for the management and support of the Graduate School of Medicine cryo-EM facility at the University of Tokyo; Y. Kamitsukasa for preparation of NLRP9 protein; K. Murakami, S. Sakurai, M. Masutani and X.-W. Zhu for initial plasmid construction; M. Kanai and S. A. Kawashima for providing HEK293T cells; and J. G. Rheinwald for providing N/TERT-1 keratinocytes. This work was supported by a Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology Grant nos 22K15046 and 20K15730 (Z.Z.), 22H02556 and 23K18211 (U.O.) and 23H00366 (T. Shimizu.); CREST, JST (grant no. JPMJCR21E4) (T. Shimizu. and K.M.); JP22H05182 (T. Shimizu. and K.M.); the Research Foundation for Pharmaceutical Sciences (Z.Z.); the Mochida Memorial Foundation for Medical and Pharmaceutical Research (Z.Z.); and the Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from the Japan Agency of Medical Research and Development (AMED) (grant no. JP21am0101115; support no. 1570, 1846, 1848).

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  1. These authors contributed equally: Zhikuan Zhang, Takuma Shibata

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Zhikuan Zhang, Akiko Fujimura, Umeharu Ohto & Toshiyuki Shimizu

  2. Division of Innate Immunity, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

    Takuma Shibata & Kensuke Miyake

  3. Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Jiro Kitaura

  4. Department of Science of Allergy and Inflammation, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Jiro Kitaura

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Contributions

Conceptualization: Z.Z. Investigation (recombinant protein generation and biochemical analysis): Z.Z. Investigation (cryo-EM analysis and image processing): Z.Z. and U.O. Investigation (cellular assay): T. Shibata, Z.Z., A.F., J.K. and K.M. Visualization: Z.Z. Validation, data curation and project administration: Z.Z. and U.O. Writing (original draft): Z.Z. and U.O. Writing (review and editing): Z.Z., U.O. and T. Shimizu. Supervision: U.O. and T. Shimizu. Funding acquisition: Z.Z., U.O. and T. Shimizu.

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Correspondence to Umeharu Ohto or Toshiyuki Shimizu.

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Extended data figures and tables

Extended Data Fig. 1 Sequence alignment of NLRP1 from various species.

Sequence alignment of human, monkey, bovine, horse, mouse (NLRP1a and NLRP1b), rat (NLRP1a), and zebrafish NLRP1 was performed using the Clustal Omega web server. Secondary structures of human NLRP1 in this study are indicated by cylinders (α helices) and arrows (β-strands) above the sequence. Residues involved in thioredoxin binding are highlighted in yellow color. Walker A, Walker B, Sensor I motifs and a conversed histidine are boxed in blue lines.

Extended Data Fig. 2 Binding assay of hNLRP1 and nucleic acids, and putative dsRNA density.

a, Binding assay of hNLRP1230–994 and dsRNAs or dsDNAs using electrophoretic mobility shift assay (EMSA). The assay was independently performed twice and representative results are shown. b, The structure of the hNLRP195–994/sfTRX complex and the cryo-EM density map (consensus map). Cryo-EM density maps are displayed in semi-transparent surface representation with hNLRP195–994/sfTRX complex at two contour levels, 0.025 (left) and 0.012 (right). Densities around HD1-HD2-LRR interface that might be attributable to the dsRNA are indicated (right).

Extended Data Fig. 3 Cryo-EM image processing and density map.

a, Cryo-EM data processing workflow of the hNLRP195–994/sfTRX complex. Representative motion-corrected micrograph, 2D class averages, 3D class-averages, gold-standard FSC curves of the final 3D reconstruction (consensus map, resolution cut-off at FSC = 0.143), the consensus map, the focused map with its focus mask of hNLRP1(NOD)/sfTRX (purple meshes), and the consensus map (colored according to the local resolution) are shown. 2D class averages were calculated using the refined particles that were used for the final reconstruction. 3D classes selected for the following analyses are indicated with red boxes. b, Representative cryo-EM density map (B-factor sharpened maps of the consensus map or the focused map if indicated) of the hNLRP195–994/sfTRX complex. The density map is segmented and displayed by domain (sfTRX, NBD, HD1, WHD, HD2 and LRR). Densities around the C32-C35 disulfide bond and ATPγS are shown. Densities of the hNLRP1/sfTRX binding interface are shown. c, Model to map (B-factor sharpened consensus map) FSC curve.

Extended Data Fig. 4 Structural comparisons of hNLRP1 and other NLRs.

a, Structural comparison of the hNLRP195–994/sfTRX complex (this study), the NLRP3/NEK7 complex (PDB: 6NPY), NOD2 (PDB: 5IRN) and NLRC4 (PDB: 4KXF). Domains of each NLR are shown in the same colors. ATPγS and ADP are shown in sphere representations. b, Superposition of the NBD-HD1 and the bound sfTRX in the structure of the hNLRP195–994/sfTRX complex onto the NLRC4 inflammasome structure (PDB: 3JBL). NLRC4 protomers are colored gray or yellow.

Extended Data Fig. 5 Structural analysis of thioredoxin binding to NLRP1.

a, Superposition of the crystal structures of oxidized (PDB: 1ERU) and reduced (PDB: 1ERT) hTRX onto the structure of the hNLRP195–994/sfTRX complex (this study). C32 and C35 of TRX are shown in stick representations. b, Inter-molecular disulfide bond formation assay between hNLRP1230–994 (sfTRX removed) and hTRX (WT, C32S, C35S or C73Y). Reducing (left) and non-reducing (right) SDS-PAGE analyses (n = 1). c, Reducing and non-reducing SDS-PAGE of the cryo-EM sample of hNLRP195–994/sfTRX (n = 1). d, Superposition of hNLRP195–994/sfTRX complex (this study) onto hTXNIP/hTRX complex (PDB: 4LL1) (left), and hTRX reductase (hTRXR)/hTRX complex (PDB: 3QFB) (right). Structures are superimposed by TRX.

Extended Data Fig. 6 Purification of MBP-hNLRP1230–994 WT and L399E proteins.

a, Gel filtration purification of MBP-hNLRP1230–994 WT (upper) and L399E (lower) proteins. Absorbance at 280 nm is shown in blue lines. b, SDS-PAGE analysis (stained with CBB) of fractions shown in (a) (n = 1).

Extended Data Fig. 7 Comparison of the NLR NBD subdomains.

a, Sequence alignment of the NBD subdomains from various NLRs (NLRP1, NLRP3, NLRP9, NLRP7, NOD2, and NLRC4). The alignment was performed using the Clustal Omega web server. Residues involved in the thioredoxin binding in the structure of hNLRP195–994/sfTRX complex are highlighted in yellow color. b, Structure comparison of the NBD subdomains of NLRP1 (this study), NOD2 (PDB: 5IRN) and NLRP3 (PDB: 7ALV). Residues involved in the thioredoxin binding in the structure of hNLRP195–994/sfTRX complex and corresponding residues for NOD2 and NLRP3 are shown in stick representations. c, StrepII tag pull-down assay using StrepII-tagged hTRX protein (6xHis tag-cleaved) and hNLRP1230–994, rNOD2ΔC, hNOD2FL, hNLRP7ΔP, hNLRP9FL and mNLRP3FL proteins. The assay was independently performed three times and representative results are shown.

Extended Data Fig. 8 Thermal shift assay of hNLRP1230–994.

Thermal shift assay of hNLRP1230–994 (sfTRX removed) with or without hTRX (1.0 eq or 2.0 eq). Means of normalized fluorescence (n = 8) versus temperature were plotted as the melting curves (left). The Tm values (n = 8) were calculated using the Boltzmann sigmodal equation and plotted with dots (right). Tm values are presented as mean values ± SEM. hNLRP1230–994 with hTRX showed a significant high Tm values. P values were determined by one-way ANOVA with two-sided Sidak’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 9 Cellular assay using N/TERT-1 keratinocyte.

a, Representative Western blot analysis of the cell lysates of N/TERT-1 keratinocytes (WT and three TRX-KO clones). b-c, The secreted level of mature IL-1β in N/TERT-1 keratinocytes (WT and three TRX-KO clones) treated with ANS (b) or poly(I:C) (c). Lipofectamine 2000 (LF) was used for intracellular delivery of poly(I:C). Bar plots are mean ± SEM. Dot plots indicate individual data points. Data are n = 3 independent biological replicates. P values were determined by two-way ANOVA with two-sided Dunnett’s multiple comparison test (control = WT). *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 10 NLRP1 inflammasome activation and regulation models.

In steady state, NLRP1 forms complex with TRX. Release of TRX from NLRP1 induces hypersensitive state, which has higher inflammasome activation. Release of TRX is possibly regulated by antioxidants, TXNIP, and TRX reductase. TRX may also play a role in regulating other NLRs.

Extended Data Table 1 Statistics for data collection and structural refinement
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Zhang, Z., Shibata, T., Fujimura, A. et al. Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome. Nature 622, 188–194 (2023). https://doi.org/10.1038/s41586-023-06532-4

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