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. Author manuscript; available in PMC: 2020 Nov 4.
Published in final edited form as: Nat Immunol. 2020 May 4;21(7):736–745. doi: 10.1038/s41590-020-0669-6

Figure 4.

Figure 4

Disulfiram covalently modifies GSDMD Cys191. (a,b) MS/MS spectra of the Cys191-containing human GSDMD peptide FSLPGATCLQGEGQGHLSQK (aa 184–103; 2057.00 Da) modified on Cys191 (red) by carbamidomethyl (an increase of 57.0214 Da) [LC retention time, 22.85 min; a triplet charged precursor ion m/z 705.6827 (mass: 2114.0481 Da; delta M 2.27 ppm) was observed] (a) or of the corresponding GSDMD peptide after GSDMD incubation with disulfiram, which was modified on Cys191 (red) by the diethyldithiocarbamate moiety of disulfiram (an increase of 147.0255 Da). [LC retention time. 28.93 min; a triplet charged precursor ion m/z 735.6802 (mass: 2204.0406 Da; delta M 0.53 ppm) was observed.] (b). Data are representative of three independent experiments. (c) Models of full-length human GSDMD in its auto-inhibited form and of the pore form of GSDMD-NT based on the corresponding structures of GSDMA3 9, 13 showing the location in yellow of Cys191, modified by compound disulfiram. GSDMD-NT in cyan; GSDMD-CT in gray. d, Dose response curve of disulfiram inhibition of liposome leakage induced by wildtype (WT), C38A or C191A human GSDMD (0.3 μM) plus caspase-11 (0.15 μM). n = 3 independent experiments. The mean ± s.e.m. is shown. e, Full-length (FL) human GSDMD and GSDMD C191S were co-expressed with caspase-11 in HEK293T cells. Cell death was determined by CytoTox96 cytotoxicity assay 20 hrs after transfection. f, FL human wildtype or C191S GSDMD were co-expressed with caspase-11 in HEK293T cells. Eight h post transfection, the indicated amount of disulfiram was added and cell death was determined by LDH release 12 hrs later. (e,f) show the mean ± s.d. of 1 representative experiment of three independent experiments performed. Comparisons in (e,f) were calculated by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, n.s., not significant.