Protein engineering reveals that gasdermin A preferentially targets mitochondrial membranes over the plasma membrane during pyroptosis

• Published in: The Journal of biological chemistry Vol. 299; no. 2; p. 102908
• Main Authors: Kondolf, Hannah C., D'Orlando, Dana A., Dubyak, George R., Abbott, Derek W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2023; American Society for Biochemistry and Molecular Biology
• Subjects: cell death; Cell Membrane - metabolism; Gasdermins; GSDMA; GSDMD; Inflammasomes - metabolism; Intracellular Signaling Peptides and Proteins - metabolism; mitochondria; Mitochondrial Membranes - metabolism; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Phosphate-Binding Proteins - metabolism; plasma membrane; Protein Engineering; protein–lipid interactions; Pyroptosis; GSDMD; HEK293T; LCIS; SSc; GSDMA; cell death; plasma membrane; mtDNA; PMA; mitochondria; TBST; PARP; OMM; pyroptosis; BSA; LDH; protein engineering; NLRP3; PI; protein–lipid interactions
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2023.102908

When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.