Extended Data Fig. 8: Structural and functional analyses of GSDMB isoforms and their relevance to cancer cell pyroptosis.
From: Structural mechanisms for regulation of GSDMB pore-forming activity
a, Close-up view of structures around exon-6 region in the GSDMB-N domain. GSDMB-N structures in the autoinhibited state and in the pore are overlaid, with exon-6 regions highlighted in wheat. Relevant structural elements are labeled and crucial residues are in sticks. b, Superimposition of IpaH7.8LRR–GSDMBiso1 and IpaH7.8LRR–GSDMBiso4 complex structures. Close-up view shows the IpaH7.8–GSDMB binding interface in the two complexes and binding residues are in sticks. c, Sequence alignments of GSDMBIsoU and GSDMBiso4. Different residues are in magenta. Exon-6 sequences are in black background. GZMA cleavage site is in blue. Green arrow marks the site for inserting the PPase sequence. Identical residues are in red background. Numbers of starting residues are on top of the sequence. d, Cleavage of GSDMBIsoU by GZMA. WT or the cleavage-site lysine mutant of GSDMBIso3 or GSDMBIsoU proteins were incubated with GZMA, followed by SDS-PAGE analyses. e, Assay of pore-forming activity in GSDMBIsoU. Liposomes were treated with indicated isoforms of purified GSDMBPP in the presence of PPase. Liposome leakage was monitored by measuring DPA chelating-induced fluorescence of released Tb3+. SDS-PAGE analyses show cleavage of GSDMBPP. f, g, Cytotoxicity of the GSDMB-N domain of indicated isoforms. f, Indicated GSDMB-N domains were transfected into 293T cells and their expression was probed by immunoblotting. LDH release-based cell death data are means (bars) of three replicates (circles). g, Indicated GSDMB-N domains under an IPTG-inducible promoter were transformed into E. coli. CFUs per transformation are shown in the logarithmic form (log10) as means (bars) of three replicates (circles). h–j, Analyses of the pyroptosis-inducing function of GSDMBIsoU. H1299 cells stably expressing an indicated isoform were electroporated with a fixed dose (h, i) or titrating amounts of GZMA (j). h, Morphological examination of cell pyroptosis. Scale bar, 25 μm. i, j, Cleavage of GSDMB was probed by immunoblotting and ATP-based cell viability, expressed as means (bars) of three replicates (circles), is shown. k, l, Analyses of relative GSDMB isoform expression in primary epithelial cells derived from human gastrointestinal tissues. Indicated cells were left untreated (NT) or stimulated with IFN-γ, TNF-α, or LPS. k, GSDMB expression was examined by immunoblotting. l, Total mRNA transcripts of GSDMB were subjected to sequencing to quantify the different isoforms. Data are representative of three (d–j) or two (k, l) independent experiments. See Supplementary Fig. 1 for gel source data.
