Abstract
Gasdermin (GSDM) family proteins, known as the executors of pyroptosis, undergo protease-mediated cleavage before inducing pyroptosis. We here discovered a form of pyroptosis mediated by full-length (FL) GSDME without proteolytic cleavage. Intense ultraviolet-C irradiation-triggered DNA damage activates nuclear PARP1, leading to extensive formation of poly(ADP-ribose) (PAR) polymers. These PAR polymers are released to the cytoplasm, where they activate PARP5 to facilitate GSDME PARylation, resulting in a conformational change in GSDME that relieves autoinhibition. Moreover, ultraviolet-C irradiation promotes cytochrome c-catalysed cardiolipin peroxidation to elevate lipid reactive oxygen species, which is then sensed by PARylated GSDME, leading to oxidative oligomerization and plasma membrane targeting of FL-GSDME for perforation, eventually inducing pyroptosis. Reagents that concurrently stimulate PARylation and oxidation of FL-GSDME, synergistically promoting pyroptotic cell death. Overall, the present findings elucidate an unreported mechanism underlying the cleavage-independent function of GSDME in executing cell death, further enriching the paradigms and understanding of FL-GSDME-mediated pyroptosis.
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Data availability
Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD051840 (ref. 61). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank C.-Q. Lei (Wuhan University, China) for kindly providing PARP5a– and PARP5b–Flag plasmids, Y. Chen (Fujian Medical University, China) for kindly providing PCDH–2×Strep–Flag vector and W.-B. Hong (School of Life Sciences, Xiamen University) for kindly elucidating the predictive architecture of the GSDME protein. We thank our colleagues at the School of Life Sciences, Xiamen University, who provided technical support, including Y. Wu, Z. Xu and C. Xie for mass spectrometry experiments and data analysis and L. Yao for electron microscopy analysis. This work was supported by the Ministry of Science and Technology of China and the National Natural Science Foundation of China (2020YFA0803403, U1905206 and 82021003 to Q.W., 32170777, U23A20450 to H.-Z.C. and 32201029 to B.Z.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Q.W. and H.-Z.C. conceived this study, generated hypotheses and designed the experiments. Q.W. and H.-Z.C. wrote, reviewed and edited the paper. B.Z., Z.-H.J., M.-R.D., Y.-L.A., L.X., L.-Z.W., Q.-T.C. and H.-Z.C. performed biological experiments and mouse experiments. C.-Q.Z. was responsible for the analysis of mass spectrometry data.
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Extended data
Extended Data Fig. 1 GSDME triggered pyroptosis in a cleavage-independent manner.
In this figure, HeLa cells were irradiated by UVC at 200 mJ cm−2 with or without pretreatment of different inhibitors for 2 h as indicated, and then cultured for 1 h to detect GSDME mitochondrial localization, 3 h to indicate GSDME oxidation, 4 h to show pyroptotic morphology and PI positive rate (%) as required, unless specially defined. a, cells were irradiated by UVC at 50 mJ cm-2, apoptosis was detected by Annv+PI−staining, PARP cleavage and DNA ladder. b, cells were irradiated by UVC at indicated doses, cell death and morphologies are shown. c, cells were cultured in DMEM medium supplemented with either 0.5% or 10% serum, pyroptosis is shown. d, cells were pretreated with different inhibitors (necrosulfonamide (NSA),10 μM; chloroquine (CQ), 20 μM; tetrathiomolybdate (TTM), 20 μM), pyroptosis was assayed. e, pyroptosis were assayed in different cancer cell lines. f, cells were transfected with different gasdermin proteins, the expression patterns are shown. g, cells were pretreated with Z-VAD (40 μM) and pyroptosis was analysed. h, the knockdown efficiency of different genes was detected. i, GSDMB, GSDMC or GSDMD was knocked down and pyroptosis was indicated. j, GSDME was knocked down and the PI images are shown. k, left, the mitochondrial fraction was prepared, and then subjected to western blotting. Right, cells were transfected with GSDME-Flag. The subcellular localization of GSDME was indicated. Tom20 was used to show the mitochondria and DAPI was used to show the nucleus. l, cells were irradiated at different doses of UVC and then cultured for 2 h, the caspase-9 and caspase-3 were detected. m, GSDMEWT and GSDMED267/270A were expressed in GSDME knockdown cells, pyroptosis was detected. Statistical data are presented as the mean ± s.e.m of (n = 3) three independent experiments. Statistical analyses were determined by Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (a, c-e, g, i), One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (b, m) and Unpaired two-tailed Student’s t-test (h). P values are indicated. All western blots were repeated at least two times and one of them is shown. Statistics source data for Extended Data Fig. 1 can be found in Statistical Source Data ED Fig. 1.
Extended Data Fig. 2 UVC induced oxidative oligomerization of GSDME.
In this figure, HeLa cells were irradiated by UVC at 200 mJ cm-2 with or without pretreatment of different inhibitors for 2 h as indicated, and then cultured for 1 h to detect lipid ROS and GSDME PM targeting, 3 h to indicate GSDME oxidation, 4 h to show pyroptotic morphology and PI positive rate (%) as required, unless specially defined. a, after pretreatment of DFO (deferoxamine) (200 μM), pyroptosis was indicated. b-d, after pretreatment of GSH (glutathione) (1 mM) or NAC (N-acetyl cysteine) (2.5 mM), the total ROS, lipid ROS (b), GSDME oxidation (c) and pyroptosis (d) were indicated. e, GSDME was knocked down and lipid ROS level was indicated. f, left, the oxidation of GSDME was detected. Right, cell culture medium was collected and filtered with 0.22 μm membrane filter, the content of GSDME was assayed. g, top, the putative motif of GSDME at Cys156 and Cys180 in different species. Bottom, different GSDME deletions and point mutants were transfected into cells, the oxidations were detected. h, cells transfected with or without GSDME-Flag were pretreated with 2-BP (2-bromopalmitate) (10 μM), GSDME palmitoylation (top), PM targeting (middle) and pyroptosis (bottom) were detected. i, cells were pretreated with FeTPPS (5 μM) or PAG (DL-propargylglycine) (5 μM), pytoptosis was indicated. j, the S-glutathionylation of GSDME and of GAPDH (positive control) were detected. k, the lysates from overexpressing GSDME cells were incubated with β-ME (10%), TCEP (50 mM) or NH2OH (200 mM) for 30 minutes. The oxidation of GSDME was detected. l, Mass spectrometry analysis confirms the presence of disulfide bonds between GSDME Cys156 and Cys180 under non-reducing condition after UVC irradiation (left). The GSDME peptide segments (light green colour) in the mass spectrum analysis were indicated (right). Statistical data are presented as the mean ± s.e.m of (n = 3) three independent experiments. Statistical analyses were determined by Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (a, b, e, h, i). P values are indicated. All western blots were repeated at least two times and one of them is shown. Statistics source data for Extended Data Fig. 2 can be found in Source Data ED Fig. 2.
Extended Data Fig. 3 UVC-induced mitochondrial fission promotes lipid ROS production.
In this figure, HeLa cells were irradiated by UVC at 200 mJ cm-2 with or without pretreatment of different inhibitors as indicated, and then cultured for 1 h to detect GSDME PM targeting, lipid ROS and mitochondrial morphology, 3 h to indicate GSDME oxidation, 4 h to indicate pyroptotic morphology and PI positive rate (%), unless specially defined. a, the levels of mRNA were detected by real-time PCR and the level of protein was detected by western blotting. b-d, FIS1, MID49 and MID51 were separately knocked down first. The pyroptosis, mitochondrial fission, and lipid ROS were indicated. e-g, cells were pretreated with Mito-Q (2 μM, 2 h), the GSDME PM targeting (e), GSDME oxidation (f) and pyroptosis induction (g) are shown. Statistical data are presented as the mean ± s.e.m of (n = 3) three independent experiments. Statistical analyses were determined by Unpaired two-tailed Student’s t-test (a) and Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (b-d, g). P values are indicated. All western blots were repeated at least two times and one of them is shown. Statistics source data for Extended Data Fig. 3 can be found in Statistical Source Data ED Fig. 3.
Extended Data Fig. 4 Cytochrome c increases lipid ROS by oxidizing cardiolipin.
In this figure, HeLa cells were irradiated by UVC at 200 mJ cm-2 with or without pretreatment of different inhibitors as indicated, and then cultured for 1 h to observe the morphologies of mitochondria, detect mito-ROS and cyt.c peroxidase activity, unless specially defined. a, cyt.c was knocked down first, and the cells were stained with Hsp60 to show the mitochondria and DAPI to show the nucleus. b, cyt.c were knocked down, the level of mito-ROS was detected. c, cells transfected with cyt.c-strep-Flag were pretreated with Fer-1 (2 μM, 2 h). Subsequently, cyt.c was pulled down by Strep-Tactin Sepharose beads. Cyt.c peroxidase activity was detected. d, the knockdown efficiency of different genes was detected. e-f, BAX and BAK were knocked down simultaneously. The cytosolic fractions were prepared and release of cyt.c was detected (e). The oxidation of cardiolipin and lipid ROS were detected (f). g, PLSCR3 was knocked down first, the oxidation of cardiolipin was detected by NAO staining (left) and the levels of lipid ROS were analysed (right). h, cells were transfected with GFP-V5-TurboID and GSDME-V5-TurboID, and then incubated with biotin for 10 minutes. After biotin was removed for 1 h, cell lysates were immunoprecipitated using streptavidin magnetic beads. The interaction between GSDME and mitochondrial outer membrane (indicated by Tom40 and Tom70) was detected. ANT1 (indicating the mitochondrial inner membrane) was used as a negative control. Statistical data are presented as the mean ± s.e.m of (n = 3) three independent experiments. Statistical analyses were determined by Unpaired two-tailed Student’s t-test (d) and Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (b, c, f, g). P values are indicated. All western blots were repeated at least two times and one of them is shown. Statistics source data for Extended Data Fig. 4 can be found in Statistical Source Data ED Fig. 4.
Extended Data Fig. 5 PARP1 facilitates UVC-induced pyroptosis.
In this figure, HeLa cells were irradiated by UVC at 200 mJ cm-2 with or without pretreatment of different inhibitors for 2 h as indicated, and then cultured for 10 minutes to detect DNA damage, total PARylation and GSDME-PARP1 interaction, 1 h to detect lipid ROS, 3 h to indicate GSDME oxidation, 4 h to show pyroptotic morphology and PI positive rate (%), unless specially defined. a, after pretreatment of Fer-1 (2 μM), the DNA damage was assessed by γH2AX expression (left) or comet assay (right). b, after pretreatment of different inhibitors, including rucaparib (20 μM), Olaparib (20 μM), Ku55933 (1 μM), AZD6738 (1 μM), and NU7441 (20 μM), the pyroptosis was analysed. c-d, After pretreatment of PDD0017273 (1 μM, 1h), the total PARylation (c) and pyroptosis (d) were detected. e-f, after pretreatment of rucaparib, the GSDME oxidation (e) and PM targeting (f) were indicated. g, efficiencies of knocking down different genes’ expression in HeLa cells. h, the GSDME PARylation was quantified. i, the fractions of cytosol and nucleus were prepared, and PARP1 or GSDME localization was indicated. j, cells were transfected with PARP1, co-IP assay was performed. k, After pretreatment of PDD0017273, the free PAR was detected. l-m, cells were pretreated with NMN (200 μM), the mito-ROS and lipid ROS (l) and pyroptosis (m) were detected. n, left, cells transfected with GSDME-Strep-Flag were irradiated by UVC at different doses, the GSDME PARylation was detected. Right, the levels of lipid ROS were detected at different UVC doses. o, after pretreatment of rucaparib or knocking down PARP1, the levels of mito-ROS and lipid ROS were detected. p, cells transfected with GSDME-Strep-Flag and its different mutations were pretreated with or without Fer-1, the GSDME PARylation was detected. Statistical data are presented as the mean ± s.e.m of (n = 3) three independent experiments. Statistical analyses were determined by Unpaired two-tailed Student’s t-test (g), Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (b, d, l, m, o) and One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (n). P values are indicated. All western blots were repeated at least two times and one of them is shown. Statistics source data for Extended Data Fig. 5 can be found in Statistical Source Data ED Fig. 5.
Extended Data Fig. 6 The PARylation of GSDME facilitates UVC-induced pyroptosis.
In this figure, HeLa cells were irradiated by UVC at 200 mJ cm-2 with or without pretreatment of different inhibitors for 2 h as indicated, and then cultured for 10 minutes to indicate GSDME-PARP5a/5b interaction and GSDME PARylation (GSDME-PAR), 1 h to indicate GSDME PM targeting, 3 h to indicate GSDME oxidation, 4 h to show pyroptotic morphology and PI positive rate (%) as required, unless specially defined. a, cells transfected with GSDME-Strep-Flag were pretreated with K756 (20 μM), the GSDME PARylation was detected. b-d, after pretreatment of K756, oxidation (b) and PM targeting (c), and pyroptosis (d) were detected. e, cells transfected with PARP5a or PARP5b were subjected to co-IP assays. f, efficiency of knocking down PARP5a/PARP5b genes. g, cells were transfected with GSDME1–220 or GSDME221–496, the GSDME-PAR was detected. h, different GSDME point mutants were transfected into cells, and the subcellular localization of GSDME was observed. i, cells were transfected with GSDME- or GSDMER238/G243A, the GSDME-PAR was detected. j, cells transfected with different plasmids were subjected to co-IP assays. k-m, GSDME- or GSDMER238/G243A were transfected into cells (k,l) or GSDME knocking down cells (m), the GSDME oxidation (k), PM targeting (l) and pyroptosis (m) were detected. Statistical data are presented as the mean ± s.e.m of (n = 3) three independent experiments. Statistical analyses were determined by Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (d) and One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (m). P values are indicated. All western blots were repeated at least two times and one of them is shown. Statistics source data for Extended Data Fig. 6 can be found in Statistical Source Data ED Fig. 6.
Extended Data Fig. 7 PARylation of GSDME induces conformational changes in GSDME.
In this figure, HeLa cells were irradiated by UVC at 200 mJ cm-2 with or without pretreatment of different inhibitors for 2 h as indicated, and then cultured for 1 h to indicate GSDME PM targeting, 4 h to indicate the pyroptotic morphology and PI positive rate (%), unless specially defined. a, top, three sites in GSDME were selected on the surface of C-terminus binding to the N terminus, and then mutated into DDDDK (Flag tag) with indicated sequence. Bottom, the positions of GSDME318–322 (blue), GSDME395–399 (red), and GSDME455–459 (purple) are depicted within the structural framework of GSDME. Three predicted structures of GSDME are generated using the AlphaFold2 algorithm. b, cells transfected with GSDME-mut2-HA were pretreated with K756 (20 μM) and then the conformational change of GSDME was detected (see Methods). c, cells were incubated PEG8000 (10%, 4 h) after UVC irradiation, the pyroptosis was detected. d, GSDMEF43/W44G was constructed in the GSDME-mut2-HA plasmid, and then transfected into cells, the conformational change of GSDME was detected. e-f, cells were transfected with GSDMEF43/W44G, the PM localization of GSDME (e) and pyroptosis (f) were indicated. g-i, in GSDME KO cells, pyroptosis (g), total PARylation (h) and lipid ROS (i) were detected. j, GSDME KO cells were transfected with different plasmids, the pyroptotic morphology was observed (j, left), and the cell death rates (%) (j, right) were detected. k, the GSDME KO cells were pretreated with rucaparib or Fer-1, the cells were cultured for an additional 12 h, and the cell death rates were determined. Statistical data are presented as the mean ± s.e.m of (n = 3) three independent experiments. Statistical analyses were determined by Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (c, e, g, i, k) and One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (f, j). P values are indicated. All western blots were repeated at least two times and one of them is shown. Statistics source data for Extended Data Fig. 7 can be found in Statistical Source Data ED Fig.7.
Extended Data Fig. 8 Combination of clinical drugs elicits FL-GSDME-mediated pyroptosis.
In this figure, HeLa cells were treated with different combination of reagents as indicated for 1 h to show GSDME PARylation, 2 h to show GSDME conformational change, 3 h to detect GSDME PM targeting, 4 h to indicate GSDME oxidation, 5 h to show pyroptotic morphology. a, different reagents were used to treat cells (5-Fluorouracil, 50 μM; Actinomycin D, 2 μg/ml; Bleomycin, 50 μM; Busulfan, 200 μM; Cisplatin, 20 μg/ml; Cisprofloxacin, 1 mM; Doxorubicin, 20 μM; Hydroxyurea, 2 mM; Mitoxantrone, 12.5 μM; Oxaliplatin, 15 μM; Paclitaxel, 10 μg/ml; Temozolomide, 200 μM; Topotecan, 20 μM; and VP-16, 100 μM) for 9 h, the cell morphologies were observed. b, RSL3 (100 nM) combined with different reagents, including MMS (methyl methanesulfonate, 1 mM), MNNG (N-methyl-N’-nitro-N’-nitrosoguanidine, 0.3 mM) and carmustine (0.25 mM)), were used to treated cells, the pyroptotic morphologies were observed. RSL3 combined with lower dose of UVC irradiation (50 mJ cm-2) was used as a positive control. c, cells transfected with GSDME-Strep-Flag were treated with RSL3 combined with different reagents or UVC irradiation (50 mJ cm-2), GSDME PARylation was determined. d, cells transfected with GSDME-mut2-HA were treated with RSL3 combined with different reagents or UVC irradiation (50 mJ cm-2), the GSDME conformation change was determined. e-f, cells were treated with RSL3 combined with different reagents or UVC irradiation (50 mJ cm-2), the GSDME oxidation (e) and GSDME PM targeting were determined (f). g, Representative flow cytometry plots illustrated the distribution of natural killer (NK) cells, perforin (PFN) and interferon-gamma (IFN-γ) expression in CD8+ T cells, along with IFN-γ expression in NK cells within the tumour microenvironment. All western blots were repeated at least two times and one of them is shown.
Extended Data Fig. 9 A novel UVC irradiation-induced model through two signalling pathways to coordinately trigger FL-GSDME-mediated pyroptosis induction.
Under UVC irradiation, DRP1/MFF triggers mitochondrial fission, which results in the upregulation of cyt.c peroxidase activity and subsequent oxidation of cardiolipin (CL), thereby increasing lipid ROS. Concurrently, nuclear PARP1-generated PAR released into the cytosol in response to DNA damage, facilitates PARP5-mediated GSDME PARylation, leading to a conformational change in GSDME. This alteration in GSDME conformation enhances its oxidation upon sensing lipid ROS to stimulate GSDME PM translocation, perforation, and ultimately pyroptosis induction.
Supplementary information
Supplementary Information
Supplementary Fig. 1.
Supplementary Table 1
This table contains oligonucleotide sequences used for shRNA-targeted mRNA.
Supplementary Table 2
This table contains primer sequences used for quantitative real-time PCR.
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Zhou, B., Jiang, Zh., Dai, Mr. et al. Full-length GSDME mediates pyroptosis independent from cleavage. Nat Cell Biol 26, 1545–1557 (2024). https://doi.org/10.1038/s41556-024-01463-2
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DOI: https://doi.org/10.1038/s41556-024-01463-2
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