Decoding gsdmd pore clustering as a novel checkpoint regulating pyroptosis

The National Institute of Allergy and Infectious Diseases, a component of the Department of Health and Human Services National Institutes of Health, awarded $267,000 to The Children's Hospital Corporation (doing business as Boston Children's Hospital) on August 17, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855).

The award funds research decoding gasdermin D (GSDMD) pore clustering as a regulatory checkpoint controlling pyroptosis, a lytic and inflammatory form of programmed cell death. The research will elucidate the organization and molecular basis of clustered GSDMD pores and define their functional role in controlling lytic cell death. The investigator hypothesizes that GSDMD palmitoylation drives formation of higher-order clustered pores on the plasma membrane, serving as a critical checkpoint that controls the transition from non-lytic cytokine release to membrane rupture during pyroptosis. Work will employ cryo-electron tomography, super-resolution DNA-PAINT, and single-molecule TIRF microscopy to define the organization and molecular mechanisms underlying GSDMD cluster-pore formation and to determine the functional significance of pore clustering. Understanding this molecular switch is intended to support development of strategies to modulate inflammation and cell death in sepsis, autoimmune diseases, and cancer.

Performance is located in Boston, Massachusetts, with an ultimate completion date of June 30, 2028. This is a project grant, classified as an assistance award to a nonprofit research institution.

Abstract

Pyroptosis is a lytic and inflammatory form of programmed cell death that plays a critical role in host defense, infection control, and cancer immunity. Gasdermin d (gsdmd) is the central effector of pyroptosis, forming plasma membrane pores that release proinflammatory cytokines such as interleukin-1Β (IL-1Β). Although the structural basis of pore formation has been elucidated, the mechanism by which gsdmd pores are regulated to determine non-lytic controlled cytokine secretion versus full lytic membrane rupture remains unknown. Understanding this molecular switch is essential for developing strategies to modulate inflammation and cell death in sepsis, autoimmune diseases, and cancer. Our preliminary study revealed a previously unrecognized regulatory mechanism: s-palmitoylation promotes the clustering of gsdmd pores, while depalmitoylation maintains them in a dispersed state. Strikingly, clustered pores correlate with plasma membrane rupture, whereas dispersed pores permit cytokine secretion without lysis. These findings suggest that palmitoylation-driven pore clustering represents a molecular checkpoint that determines the lytic outcome of pyroptosis. The overall objective of this program is to elucidate the organization and molecular basis of clustered gsdmd pores and to define its functional role in controlling lytic cell death. We hypothesize that gsdmd palmitoylation drives the formation of higher-order clustered pores on the plasma membrane, serving as a critical checkpoint controlling the transition from non-lytic cytokine release to catastrophic membrane rupture during pyroptosis. To test this hypothesis, we will define the organization and molecular mechanisms underlying gsdmd cluster-pore formation using cryo-electron tomography, super-resolution DNA-paint, and single-molecule tirf microscopy. We will also determine the functional significance of pore clustering in regulating cytokine secretion and membrane rupture using palmitoylation-deficient gsdmd mutants and dual IL-1Β/LDH reporter assays. This program will develop a new conceptual framework explaining how gsdmd activity is tuned between cytokine release and membrane rupture. IT will introduce the novel concept that gsdmd pores form higher-order clusters that act as a mechanistic checkpoint controlling lytic cell death. These results will establish gsdmd pore clustering as a druggable regulatory layer of inflammation, providing a foundation for therapeutic strategies against inflammatory and cancer- related diseases.