Fig. 4: Methylcobalamin coordinates to Cys180 to block GSDME cleavage.
From: Methylcobalamin protects against liver failure via engaging gasdermin E
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 Kd 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 Kd 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.
