Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells

Abstract

Gasdermin (GSDM)-mediated pyroptosis is functionally involved in multiple diseases, but Gasdermin-B (GSDMB) exhibit cell death-dependent and independent activities in several pathologies including cancer. When the GSDMB pore-forming N-terminal domain is released by Granzyme-A cleavage, it provokes cancer cell death, but uncleaved GSDMB promotes multiple pro-tumoral effects (invasion, metastasis, and drug resistance). To uncover the mechanisms of GSDMB pyroptosis, here we determined the GSDMB regions essential for cell death and described for the first time a differential role of the four translated GSDMB isoforms (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Accordingly, we here prove that exon 6 translation is essential for GSDMB mediated pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in breast carcinomas the expression of GSDMB2, and not exon 6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Mechanistically, we show that GSDMB N-terminal constructs containing exon-6 provoke cell membrane lysis and a concomitant mitochondrial damage. Moreover, we have identified specific residues within exon 6 and other regions of the N-terminal domain that are important for GSDMB-triggered cell death as well as for mitochondrial impairment. Additionally, we demonstrated that GSDMB cleavage by specific proteases (Granzyme-A, Neutrophil Elastase and caspases) have different effects on pyroptosis regulation. Thus, immunocyte-derived Granzyme-A can cleave all GSDMB isoforms, but in only those containing exon 6, this processing results in pyroptosis induction. By contrast, the cleavage of GSDMB isoforms by Neutrophil Elastase or caspases produces short N-terminal fragments with no cytotoxic activity, thus suggesting that these proteases act as inhibitory mechanisms of pyroptosis. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer or other pathologies and for the future design of GSDMB-targeted therapies.

Structural basis for GSDMB pore formation and its targeting by IpaH7.8

“…By contrast, isoform 3 with a truncated interdomain linker would probably preserve the oligomerization interface but lacks the basic cluster for lipid binding, and isoform 2 lacks the entire interdomain linker for oligomerization and lipid binding. A recent study showing that GSDMB isoforms without exon 6, which encodes the canonical sequence in the interdomain linker, did not induce pyroptosis strongly supports our model 3 .…”

“…In addition to bacterial infection, GSDMB is associated with various cancers. One recent study indicated that expression of non-pyroptotic isoform 2 was higher than that of other isoforms in patients with breast cancer 3 . Upregulation of isoforms 2 and 3 may promote tumorigenesis and metastasis, leading to poor overall patient survival, whereas high expression of pyroptotic isoform 4 has the opposite effect 3 .…”

“…7c). Rather, the linker may play roles in regulation of GSDMB pore-forming activity 3 . Previous studies have shown that GSDMB isoforms 4 and 6 containing the canonical sequence in the interdomain linker showed strong membrane-permeabilizing activities both in vitro and in cells 5,7 , whereas isoform 2, which lacks the canonical sequence, does not have pyroptotic activity 3 .…”

“…Rather, the linker may play roles in regulation of GSDMB pore-forming activity 3 . Previous studies have shown that GSDMB isoforms 4 and 6 containing the canonical sequence in the interdomain linker showed strong membrane-permeabilizing activities both in vitro and in cells 5,7 , whereas isoform 2, which lacks the canonical sequence, does not have pyroptotic activity 3 . Surprisingly, isoform 1, also containing the canonical sequence in its interdomain linker, does not induce pyroptotic cell death; rather, it targets bacterial membranes to kill bacteria 4 .…”

“…Gasdermins (GSDMs) are pore-forming proteins that play critical roles in host defence through pyroptosis 1,2 . Among GSDMs, GSDMB is unique owing to its distinct lipidbinding profile and a lack of consensus on its pyroptotic potential [3][4][5][6][7] . Recently, GSDMB was shown to exhibit direct bactericidal activity through its pore-forming activity 4 .…”