Mechanisms of gasdermin-dependent epithelial function in inflammatory bowel disease.

Case Western Reserve University, Cleveland OH

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Abstract

Increasing evidence indicates that inflammatory bowel disease (IBD) is considered an intestinal barrier disorder, whereby epithelial dysfunction is central to disease pathogenesis. Single nucleotide polymorphisms (SNPs) in gasdermin B (GSDMB), a gene known to be predominantly expressed in the epithelium of the GI tract, are associated with increased susceptibility to IBD. GSDMB belongs to a family of structurally-related pore-forming proteins, i.e., gasdermins (GSDMs), primarily known for their central role in programmed cell death, or pyroptosis. Indeed, GSDMB can be cleaved by Granzyme A (GzmA), whereby the released N-terminal domain (GSDMB- NTD) oligomerizes to form pores in the plasma membrane, thus mediating pyroptosis. GSDMB-NTD also exhibits direct microbicidal activity, targeting gram-negative bacteria, while sparing host epithelial cells. Our group showed that in its full-length form, GSDMB exerts crucial, non-lytic functions within intestinal epithelial cells (IECs) to control mucosal wound healing, and importantly, GSDMB is not only increased in IBD patients relative to healthy controls, but IBD-associated mutant GSDMB (GSDMBMut) confers dysregulated IEC function(s). Numerous GSDMB protein isoforms have been described, and were thought to account for the aforementioned functional differences. Several papers published in 2023 reported the structural and biochemical analyses of GSDMB, and came to the consensus that a singular exon within its interdomain linker region (i.e., Exon 6) dictates the pore-forming, pyroptotic activity of GSDMB upon GzmA-mediated cleavage and activation. Despite these advances, there remains a significant knowledge gap in resolving the relevance and significance of GSDMB isoforms during health vs. disease states. Our preliminary findings indicate a GI-specific pattern of GSDMB transcript expression in gut mucosal biopsies with a marked shift in IBD patients. Genomic analysis further demonstrates that the rs11078926 GSDMB SNP not only alters splicing and isoform expression, but may potentially contribute to IBD pathophysiology. Furthermore, much attention has recently focused on GSDM regulation by post-translational modifications, specifically S-palmitoylation, leading to differential cellular function(s). Our preliminary data also suggest that cysteine modifications, but mainly by S-nitrosylation, plays a critical role in regulating GSDMB-driven IEC activities. Taken together, the central hypothesis of this proposal is that GSDMB isoforms are differentially expressed in IECs of control vs. IBD patients, whereby normal vs. defective epithelial function is determined in an isoform-dependent fashion and impacted by carriage of GSDMBMut variants; furthermore, IEC-derived GSDMB is differentially regulated by S-nitrosylation that subsequently impacts GSDMB-dependent IEC activities. The overarching goal of the present proposal is to identify and characterize clinically-relevant GSDMB isoform(s) and determine how their regulation can differentially affect epithelial function. This information will not only provide insights into the role of GSDMB in IBD pathophysiology, but aid in the design of novel therapeutics targeting GSDMB-dependent IEC function.