Caspase-1 activates gasdermin A in non-mammals

Abstract

Gasdermins oligomerize to form pores in the cell membrane, causing regulated lytic cell death called pyroptosis. Mammals encode five gasdermins that can trigger pyroptosis: GSDMA, B, C, D, and E. Caspase and granzyme proteases cleave the linker regions of and activate GSDMB, C, D, and E, but no endogenous activation pathways are yet known for GSDMA. Here, we perform a comprehensive evolutionary analysis of the gasdermin family. A gene duplication of GSDMA in the common ancestor of caecilian amphibians, reptiles and birds gave rise to GSDMA-D in mammals. Uniquely in our tree, amphibian, reptile and bird GSDMA group in a separate clade than mammal GSDMA. Remarkably, GSDMA in numerous bird species contain caspase-1 cleavage sites like YVAD or FASD in the linker. We show that GSDMA from birds, amphibians, and reptiles are all cleaved by caspase-1. Thus, GSDMA was originally cleaved by the host-encoded protease caspase-1. In mammals the caspase-1 cleavage site in GSDMA is disrupted; instead, a new protein, GSDMD, is the target of caspase-1. Mammal caspase-1 uses exosite interactions with the GSDMD C-terminal domain to confer the specificity of this interaction, whereas we show that bird caspase-1 uses a stereotypical tetrapeptide sequence to confer specificity for bird GSDMA. Our results reveal an evolutionarily stable association between caspase-1 and the gasdermin family, albeit a shifting one. Caspase-1 repeatedly changes its target gasdermin over evolutionary time at speciation junctures, initially cleaving GSDME in fish, then GSDMA in amphibians/reptiles/birds, and finally GSDMD in mammals.

Figure 1.. Phylogenetic analysis of GSDM proteins.

Time-calibrated maximum clade credibility coalescent Bayesian phylogenetic tree of all protein sequences in selected superphyla by HMM identification method. Tree was visualized using iTOL v6 (Letunic and Bork, 2021). figure supplement 1. Tree with node tips labeled. figure supplement 2. Tree with node tips labeled and posterior probabilities displayed. figure supplement 3. Maximum likelihood tree. figure supplement 4. Synteny of GSDMA in animals. source data 1. Tree in .tre format. source data 2. Alignment file of all sequences in nexus format.

Figure 2.. Analysis of bird GSDMA sequences suggests caspase-1 cleavage.

(A.) Cartoons used in this study to represent organisms and their phylogenetic classification. Note that caecilian amphibians morphologically are similar to snakes in their lack of legs and elongated body, however, this is an example of convergent evolution as caecilians are amphibians and are thus more closely related to frogs and salamanders than snakes. (B.) Sequence logo plots derived from the tetrapeptides found in amphibian, bird and reptile GSDMA organized by evolutionary clades. Importantly, “Other birds” is not a monophyletic group. Sequence logos display abundance of amino acids in each position before an aspartic acid in the linker region of each GSDMA protein using WebLogo (Crooks et al., 2004). When more than one aspartic acids were present, multiple tetrapeptides were listed. (C.) Alignment of selected amphibian, reptile, bird and mammal GSDM linker regions by MUSCLE. Linker defined as region between β11 and α5 by SWISS-MODEL. Outlined in a red box are tetrapeptide sequences that precede caspase-1 cleavage sites for mammal, or show similarity in amphibian, reptile and bird sequences. The canonical caspase-1 cleavage site YVAD is emphasized with an arrowhead. “*” denotes cleavage sites of SpeB for human and mouse GSDMA. Amino acids colorized using Clustal color scheme.

Figure 3.. Chicken caspase-1 cleaves chicken GSDMA and mammal GSDMD.

(A.) Normalized relative GSDMA abundance by qRT-PCR using RNA derived from various Gallus gallus domesticus tissues. The cecal tonsils are a lymphoid aggregate tissue found in the chicken GI tract, akin to Peyer’s patches in mammals. ****, p<0.0001 by 2 way ANOVA with Tukey’s multiple comparisons correction. (B.) Diagram of constructs used in this study. Note that CARD domain of caspase-1 is not present. (C.) FLAG blot of HEK293T/17 lysates after co-transfection of chicken GSDMA and caspase-1 after addition of DMSO or AP20187 dimerizer. (D.) FLAG blot of time course of dimerizer addition. Samples were harvested at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4.5, and 6 hours after addition of AP20187 dimerizer. (E-H.) Caspase-1 from chicken, human and mouse cleave bird GSMDA and mammal GSDMD. FLAG blot of HEK293T/17 lysates after co-transfection with indicated caspase-1 and GSDM constructs. P1 refers to the fourth reside of the tetrapeptide sequence, which is cleaved by caspase-1. P1 mutants are chicken D244A, crow D243A, human D275A, and mouse D276A. (I.) FLAG blot of HEK293T/17 lysates after co-transfection with indicated gasdermin and caspase-1. Caspase-1 from chicken, human and mouse cleave human GSDMD but not human GSMDA. (J.) FLAG blot of HEK293T/17 lysates after co-transfection with indicated gasdermin and caspase-9. P1 mutant of chicken GSDME is ₂₇₀DAVD₂₇₃ to ₂₇₀DAVA₂₇₃. figure supplement 1. Tissue expression of chicken GSDME and CASP1 figure supplement 2. Cleavage of crow GSDMA with recombinant Human CASP1 and CASP3.

Figure 4.. Cleavage of chicken GSDMA leads to cell lysis.

(A.) Measurement of GFP fluorescence in HEK293T/17 cells co-transfected with GFP and either full length (FL) chicken GSDMA or its N-terminal (NT) pore-forming domain after 48 hours. Each point represents a separate transfection experiment. ****, p<0.0001 by student’s t-test. (B.) Cell lysis measured by LDH release in HEK293T/17 cells co-transfected with chicken caspase-1 and either WT or D244A chicken GSDMA. Gasdermin constructs in this study do not encode the 3xFLAG domain. Each point represents a separate transfection experiment. **, p<0.01; ****, p<0.0001 by 2 way ANOVA with Tukey’s multiple comparisons correction. (C.) Images of HeLa cells after transfection with the N-terminal domain of chicken GSDMA. Transfected cells display classic pyroptotic ballooning. SYTOX green staining of the nucleus indicates permeabilization of cell membranes.

Figure 5.. Chicken GSDMA requires a compatible tetrapeptide for activation.

(A.) AlphaFold predictions of the structure of α7′ and α8 helices of human GSDMD and chicken GSMDA. α8 helices were aligned in Pymol. The human interhelix loop is indicated by the arrow. (B.) Gasdermin C-terminus AlphaFold predictions visualized with surface projection. Structures are aligned at the α8 helix. The arrow indicates the human interhelix loop and the space where human caspase-1 interacts with the C-terminal domain of human GSDMD (PDB: 6KN0, (Wang et al., 2020a)). (C.) Alignment of α7′-interhelix-α8 helix regions of human GSDMD (residues 347–380) and chicken GSDMA (residues 314–347). Residues colored with the Kyte and Doolittle scale with red indicating hydrophobic and blue indicating hydrophilic. “*” indicates residues determined to be critical for the hydrophobic interaction between human GSDMD and caspase-1. (D.) FLAG blot of HEK293T/17 cell lysates after co-transfection with either chicken or human caspase-1 and their cognate GSDM with the tetrapeptide as WT, YVAD, or AAAD. (E.) Quantification of the intensity of the N-terminal band as a ratio of the N-terminal fragment over the full length, with the greatest ratio in a given experiment set to 100% efficiency. Each point is a blot from a separate transfection. a, p<0.05; b, p<0.0001; c, p<0.01 by 1 way ANOVA with Tukey’s multiple comparisons correction. source data 1. AlphaFold prediction files in .pdb format.

Figure 6.. GSDMA from non-mammals are cleaved by caspase-1 and SpeB.

(A-C.) FLAG blot of HEK293T/17 lysates after co-transfection of A. mississippiensis, A. carolinensis, or M. unicolor GSDMA and caspase-1 from chicken, human and mouse. M. unicolor is an amphibian despite sharing morphological similarity to a snake. (D.) FLAG blot of HEK293T/17 lysates after co-transfection of platypus GSDMD or GSDMA with caspase-1 from chicken, human and mouse. (E.) FLAG blot of HEK293T/17 lysates after incubation with SpeB. Total protein captured by Ponceau staining for this blot. figure supplement 1. Amphibian expression of CASP1, GSDMA and GSDME.

Figure 7.. Caspase-1 targets different gasdermins throughout evolution.

Phylogenetic tree indicating which GSDM caspase-1 cleaves in various clades of life. Branch lengths are not to scale. Coral, mollusks, hydras and other invertebrates do not encode caspase-1 and cleave GSDM_in with caspase-3. Coral and mollusks are indicated in this diagram. We did not identify any gasdermins in 3,030 genomes from Ecdysozoa, the clade of which D. melanogaster and C. elegans are members. *Some teleost fish cleave GSDME with both caspase-1 and caspase-3, however, other teleost fish, like the zebrafish, cleave GSDMEb with caspase-1 and GSDMEa with caspase-3, gasdermins that arose after a whole genome duplication event in teleost fish. Frogs and salamanders do not encode an identifiable GSDMA, but their GSDME contains prototypical caspase-3 cleavage sites. In mammals, GSDMD is cleaved by caspase-1 and GSDME by caspase-3. In caecilian amphibians—which are morphologically similar to snakes—birds, and reptiles, GSDMA is cleaved by caspase-1. We and others have confirmed that GSDME in birds is cleaved by caspase-3 (Fig 3J, Fig 3-S2), and amphibian and reptile GSDME encodes prototypical caspase-3 cleavage sites.