Methylcobalamin protects against liver failure via engaging gasdermin E

Abstract

Gasdermin E (GSDME) is a pyroptotic cell death effector and a promising target for pyroptotic tissue injury. Here we perform high-throughput screening and demonstrate that methylcobalamin (MeCbl), an endogenous coenzyme form of vitamin B12, is a specific GSDME inhibitor and highly effective against cholestatic liver failure. MeCbl specifically blocks GSDME cleavage by directly binding with GSDME. In cholestasis-, cisplatin- or concanavalin A (Con A)-induced male mouse models, MeCbl significantly suppresses liver transaminase activities and inflammation, alleviates hepatocyte death, and reduces mortality of mice by blocking GSDME cleavage. The conserved Cys180 residue in GSDME is essential for caspase-3/GzmB recognition. MeCbl in base-off conformation coordinates to Cys180 to prevent caspase-3/GzmB-GSDME interactions and thereby GSDME-mediated pyroptosis. In summary, our study discovers MeCbl as a specific GSDME inhibitor that is promisingly to be developed as an effective drug against cholestatic liver failure, and other GSDME triggered sterile inflammation and/or organ failure.

Fig. 1. Methylcobalamin is a specific and direct inhibitor of GSDME.

a Procedure of terbium (Tb³⁺)/dipicolinic acid (DPA) fluorescence liposome leakage assay. b Percentage of inhibition on 829 compounds, assayed at 100 μM. Cutoff was 70% inhibition. Dimethyl fumarate (DMF) was used as a positive control. Each point is an individual inhibitor (n = 1). c LDH release assay for cytotoxicity of HepG2 cells pretreated with 9 screening hits at 100 μM before challenged with TNFα/cycloheximide (CHX) for 16 h to induce GSDME-mediated pyroptosis. d Chemical structure of compound 19A11 (methylcobalamin, MeCbl). e 25 ~ 100 μM MeCbl or DMF were treated with 0.2 μM recombinant active-caspase-3 for activity assay. Z-DEVD-FMK was used as positive control. f MST measurements of the binding of 0.2 μM recombinant caspase-3, GSDME, GSDMD with MeCbl or positive control. Values of K_d are listed in the graphs. Data are mean ± SEM (n = 3) of at least two independent experiments for bar charts. Analysis was done using one-way ANOVA (c, e). p < 0.05 is considered significant and p values are provided in graphs, n.s = nonsignificant. All groups were compared to indicated vehicle. Source data are provided as Source Data file.

Fig. 2. Methylcobalamin inhibits liposome leakage by preventing GSDME cleavage.

a, b Dose response of MeCbl or DMF on liposome leakage induced by cleaved human GSDME (a) or human GSDMD (b). c, Time course of liposome leakage in the presence of 1 μM MeCbl or DMF. d, e Electron microscopy images of nanodiscs that were pretreated with inhibitors as indicated, recombinant active caspase-3 and GSDME were then added for pore formation (d). Scale bar= 200 nm. Quantitative data of the long diameter of 10 liposomes of each group (e), each point is an individual liposome. f Representative immunoblot of GSDME pretreated with inhibitors as indicated, and then incubated with recombinant active caspase-3 for GSDME activation analysis. g Dose response of MeCbl on liposome leakage induced by precleaved human GSDME. h–j Dose response of MeCbl on liposome leakage induced by human GSDME-NT cleaved by granzyme B (GzmB) (h), representative immunoblot of recombinant GSDME pretreated with inhibitors and then incubated with recombinant active GzmB (i), 25 ~ 100 μM MeCbl was pretreated with 0.4 μM recombinant active GzmB for activity assay. 3,4-dichloroisocoumarin (DCI) was used as positive control (j). Data are mean ± SEM of at least two independent experiments for bar and line charts, n = 3 other than quantitative data of the long diameter of liposomes (e, n = 10). Blots and micrographs are representative of three independent experiments. Analysis was done using one-way ANOVA (e, j) or two-way ANOVA (a–c). p < 0.05 is considered significant to indicated group and p values are provided in graphs, n.s = nonsignificant compared to vehicle. Source data are provided as Source Data file.

Fig. 3. Methylcobalamin specifically inhibits GSDME-mediated pyroptosis.

a, c, d, e HepG2 were pretreated with indicated concentrations of each compound for 2 h before adding 200 μM deoxycholic acid (DCA) for 4 h, the cytotoxicity of HepG2 cells was determined by LDH release assay (a). Pyroptosis was measured by SYTOX green uptake in the presence of 20 μM MeCbl or DMF (c). Representative immunoblotting analysis of caspase-3 and GSDME in HepG2 (d), and caspase-3 activity was assessed with substrate Ac-DEVD-pNA (e). b Bone marrow derived macrophages (BMDMs) were pretreated with indicated concentrations of MeCbl for 2 h before challenged with LPS transfection by Fugene HD for GSDMD-mediated pyroptosis. Cytotoxicity was determined by LDH release assay. f Changes in HepG2 cell morphology were observed with a microscope (scale bar= 10 μm). g Representative images of GSDME localization in HepG2 cells treated as indicated by confocal microscopy (scale bar= 5 μm). h, i Mice primary hepatocytes were pretreated with indicated concentrations of each compound for 2 h before challenged with 40 nM perforin (PFR) and 0.5 μM GzmB simultaneously for 24 h to activate GSDME-mediated pyroptosis, the cytotoxicity of hepatocytes was determined by LDH release assay (h). Pyroptosis was measured by SYTOX green uptake in the presence of 20 μM MeCbl or DMF (i). j Effect of MeCbl on PFR+GzmB-induced LDH release in hepatocytes pretreated with Z-DEVD-FMK (20 μM). k, m WT, Gsdmd^-/-, Gsdme^-/- hepatocytes were pretreated with indicated inhibitors for 2 h before challenged with Fugene HD/LPS for 16 h (k) or 200 μM DCA for 4 h (m), cytotoxicity was determined by LDH release assay. l, n Representative immunoblotting analysis of GSDMD and GSDME in hepatocytes. Data are mean ± SEM (n = 3) of at least two independent experiments for bar and line charts. Blots and micrographs are representative of three independent experiments. Analysis was done using one-way ANOVA (e) or two-way ANOVA (a–c, h–k, m). p < 0.05 is considered significant to the indicated group and p values are provided in graphs, n.s = nonsignificant compared to vehicle (e) or indicated group (k, m). Source data are provided as Source Data file.

Fig. 4. Methylcobalamin coordinates to Cys180 to block GSDME cleavage.

a, b Representative mass spectrometry spectra of MeCbl coordinated to Cys180 in human GSDME peptide KCGGIVGIQTK (b), GSDME incubated with ddH₂O was used as negative control (a). The binding mode of MeCbl (base-off conformation) to GSDME was shown in graph b. c LDH release assay of Gsdme^-/- hepatocytes overexpressed with indicated GSDME plasmids. Cells were pretreated with 20 μM MeCbl, then challenged with 200 μM DCA for 4 h. d Representative immunoblotting analysis of GSDME cleavage in Gsdme^-/- hepatocytes overexpressed with indicated GSDME plasmids and challenged by DCA. e Co-immunoprecipitation (Co-IP) analysis of overexpressed GSDME with cleaved-caspase-3 in Gsdme^-/- hepatocytes treated with 200 μM DCA for 1 h. f MST measurements of the binding of 0.2 μM recombinant WT and cysteine mutant GSDME with recombinant human caspase-3. Values of K_d are listed in the graphs. g LDH release assay of Gsdme^-/- hepatocytes overexpressed with indicated GSDME-NT plasmids. h Co-IP analysis of endogenous GSDME with cleaved-caspase-3 in WT hepatocytes pretreated with 20 μM MeCbl or DMF, then cells were challenged with 200 μM DCA for 1 h. i MST measurements of the binding of 0.2 μM recombinant WT and cysteine mutant GSDME with MeCbl. Values of K_d are listed in the graphs. j Dose response of MeCbl on liposome leakage induced by WT and cysteine mutant GSDME. Data are mean ± SEM (n = 3) of at least two independent experiments for bar and line charts. Blots are representative of three independent experiments. Analysis was done using two-way ANOVA. p < 0.05 is considered significant to the indicated group and p values are provided in graphs, n.s = nonsignificant compared to vehicle (g) or indicated group (c). Source data are provided as Source Data file.

Fig. 5. Methylcobalamin protects against GSDME-initiated liver failure.

5 dpf zebrafish larvaes were pretreated with 20 μM MeCbl before immersed in 400 μM DCA for 48 h. a–c SYTOX fluorescence probe and immunoblotting analysis were used to investigate the protective effects of MeCbl on DCA-induced systemic and liver injury (liver region was indicated with red or yellow circle, scale bar=250 μm) in zebrafish, n = 6. d Survival analysis of zebrafish after DCA immersion, n = 60. e Sequence alignment of GSDME from human, mouse and zebrafish. In addition, mice were performed with bile duct ligation (BDL) surgery to induce GSDME-mediated acute liver failure. MeCbl (10 mg/kg), DMF (50 mg/kg) were given intragastrically to mice daily before euthanized. f–j Serum ALT, AST, IL-1α, IL-1β, HMGB1 levels in WT and Gsdme^-/- mice, n = 6. k Representative immunoblots of caspase-3 and GSDME in the livers of the mice. l Survival analysis of WT and Gsdme^-/- mice after BDL and treated as indicated, n = 10. m, n Representative H&E sections of the liver (m) (scale bar = 50 μm). Damaged area was traced with black dash line and analyzed using Image J software (n, n = 6 representative section of six independent mice samples). Data are mean ± SEM of at least two independent experiments. Each point is an individual zebrafish larvae or mouse, number is indicated, n = 6-60 per group. Blots are representative data of six independent samples (c, k). Analysis was done using two-tailed unpaired Student’s t test (a) or two-way ANOVA (f–j, n). Log-rank (Mantel-Cox) test was used for survival analysis (d, l). p < 0.05 is considered significant to indicated group and p values are provided in graphs, n.s=nonsignificant. Source data are provided as Source Data file.

Fig. 6. Cys180 mutant in GSDME impairs methylcobalamin intervention in vivo.

GSDME Cys mutant plasmids constructed in adenovirus vector were injected into Gsdme^-/- mice via tail vein, a GSDME expression in mice liver was detected with immunoblotting analysis. Mice were orally given 10 mg/kg MeCbl daily after BDL surgery. b–e Serum ALT, AST, IL-1α and IL-1β levels in serum 5 days after BDL/sham, n = 6. f Co-IP analysis of overexpressed GSDME with cleaved-caspase-3 in Gsdme^-/- liver. g, h Representative H&E sections of the liver (g) (scale bar = 50 μm). Damaged area was traced with black dash line and analyzed using Image J software (h, n = 6 representative section of six independent mice samples). Data are mean ± SEM of at least two independent experiments. Each point is an individual mouse, number is indicated, n = 6–10 mice/group. Blots are representative data of six independent samples (a, f). Analysis was done using two-way ANOVA (b–e, h). p < 0.05 is considered significant to indicated group and p values are provided in graphs, n.s=nonsignificant. Source data are provided as Source Data file.