Structural and functional insights into GSDMB isoforms complex roles in pathogenesis

Abstract

SHADSGasdermins (GSDMs) have garnered significant scientific interest due to their protective and detrimental roles in innate immunity, host defense, inflammation, and cancer alongside with other pathologies. While GSDMs are mostly recognized as key effectors of a lytic type of pro-inflammatory cell death known as pyroptosis, they do also take part in other cell death processes (NETosis, secondary necrosis, or apoptosis) and exhibit cell-death independent functions depending on the cellular context. Among GSDMs, Gasdermin B (GSDMB) pyroptotic capacity has been a subject of conflicting findings in scientific literature even when its processing, and subsequent activation, by Granzyme A (GZMA) was decoded. Nevertheless, recent groundbreaking publications have shed light on the crucial role of alternative splicing in determining the pyroptotic capacity of GSDMB isoforms, which depends on the presence of exon 6-derived elements. This comprehensive review pays attention to the relevant structural differences among recently crystalized GSDMB isoforms. As a novelty, the structural aspects governing GSDMB isoform susceptibility to GZMA-mediated activation have been investigated. By elucidating the complex roles of GSDMB isoforms, this review aims to deepen the understanding of this multifunctional player and its potential implications in disease pathogenesis and therapeutic interventions. [Figure: see text].

Keywords: Gasdermin; alternative splicing; cancer; cell death; inflammatory diseases; pyroptosis.

Figure 1.

Common model for GSDM-mediated pyroptosis and comparison of GSDMB isoforms. a) main steps in the general mechanism of pyroptotic cell-death mediated by Gasdermins (GSDMs). In basal conditions, GSDMs remain in the cytoplasm in a closed conformation: NTD inhibited by CTD (NTD: amino terminal and CTD: carboxy terminal domains). In response to different stimuli, specific proteases cleave the GSDM interdomain region, releasing the NTD. After structural reorganization, NTDs, now active, form large transmembrane pores, typically consisting of 23–27 monomers, releasing pro-inflammatory molecules (IL-1β/IL-18) and allowing H₂0 and ion influx. Osmotic shock causes cellular ballooning and membrane rupture, secreting DAMPs that together with cytokines activate the immune system. b) scheme of aligned amino acid (aa) sequences from GSDMB functional isoforms highlighting the NTD (green), CTD (gray) and the unique linker regions. The differences in residues from exon 6 (magenta) and 7 (ochre) are indicated. Likewise, both the aa insertion from the non-canonical 411aa isoform and the Grazyme A (GZMA) cleavage site (at K244 within exon 8) are highlighted in purple and blue respectively. Each feature in the scheme is accompanied by its corresponding aa number, which varies for each represented isoform. On the right it has been indicated the pyroptotic capacity. c) crystal structures of full-length GSDMB isoforms − 1 (PDB-ID: 8GTK), −4 (PDBid:8GTJ) and 411aa (PDBid:8EFP). NTD (green), CTD (gray) and exon 6 residues (magenta).

Figure 2.

GSDMB isoform-3 structure in complex with GZMA. Model of the general structure of GSDMB isoform-3 based on 8GKT crystal (isoform-1) [11], depicted in two forms: a) ribbon diagram showing structural domains and b) protein surface representation. NTD in green, CTD in gray, with exons 6 and 7 dyed in magenta and ochre, respectively. Position of GZMA cleavage residue K244 is indicated. c) model for the interaction between GSDMB and GZMA refined through 100ns of molecular dynamics (MD) simulations and a d) detailed view of the interface between modeled GSDMB isoform-3 in complex with GZMA. GZMA surface is colored according to electrostatic potentials, with the negatively charged pocket (red) surrounding the GSDMB residue K244. e) stability analysis in terms of root-mean-square deviation (rmsd in angstroms -Å-) of the interaction between GZMA and the distinct GSDMB variants during 100ns of MD simulation. f) Contact-based prediction of binding affinity in GSDMB-GZMA complexes for the diverse GSDMB isoforms. In protein complex formation, lower calculated free energy (ΔG, kcal mol-1) and dissociation constant (kd, (M) at 25°C) correspond to higher protein affinity.