Gasdermin D pore-forming activity is redox-sensitive

Devant, Pascal; Boršić, Elvira; Ngwa, Elsy M.; Xiao, Haopeng; Chouchani, Edward T.; Thiagarajah, Jay R.; Hafner-Bratkovič, Iva; Evavold, Charles L.; Kagan, Jonathan C.

doi:10.1016/j.celrep.2023.112008

Cited by 69 publications

(33 citation statements)

References 41 publications

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“…Our studies reveal a specific effect on palmitoylation downstream of the rather non-specific ROS, which makes biological sense. They affirm the critical point that oxidative stress directly promotes GSDMD palmitoylation, independent of the upstream inflammasome pathway and GSDMD cleavage, and explains the key role of ROS in GSDND-NT pore formation (22,23).…”

Section: Discussionsupporting

confidence: 55%

“…The Cys191/192 residue is also the target for other modifications including oxidation (23), and the target of multiple small molecules that inhibit GSDMD functions, including necrosulfonamide (NSA), disulfiram (DSF) and dimethyl fumarate (DMF) (43)(44)(45). It is templating to speculate that the mechanism of inhibition by these small molecules in cells may be related to competition with GSDMD palmitoylation at the Cys191/192 site.…”

Section: Discussionmentioning

confidence: 99%

“…Reactive oxygen species (ROS) are composed of a number of heterogeneous chemical entities that induce oxidative stress (18) and have long been recognized as an important factor in NLRP3 activation and inflammasome formation (19)(20)(21). Intriguing, recent studies also implicated a role of ROS in GSDMD-NT pore formation independent of upstream processes (22,23), but the mechanism of this role remained unclear.…”

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confidence: 99%

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ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

Healy

David

et al. 2023

Preprint

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Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Here we report the surprising finding that GSDMD cleavage is not sufficient for its pore formation. Instead, GSDMD is lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation, suggesting that palmitoylation licenses GSDMD activation. Treatment by the palmitoylation inhibitor 2-bromopalmitate and alanine mutation of Cys191 abrogate GSDMD membrane localization, cytokine secretion, and cell death, without affecting GSDMD cleavage. Because palmitoylation is formed by a reversible thioester bond sensitive to free thiols, we tested if GSDMD palmitoylation is regulated by cellular redox state. Lipopolysaccharide (LPS) mildly and LPS plus the NLRP3 inflammasome activator nigericin markedly elevate reactive oxygen species (ROS) and GSDMD palmitoylation, suggesting that these two processes are coupled. Manipulation of cellular ROS by its activators and quenchers augment and abolish, respectively, GSDMD palmitoylation, GSDMD pore formation and cell death. We discover that zDHHC5 and zDHHC9 are the major palmitoyl transferases that mediate GSDMD palmitoylation, and when cleaved, recombinant and partly palmitoylated GSDMD is 10-fold more active in pore formation than bacterially expressed, unpalmitoylated GSDMD, evidenced by liposome leakage assay. Finally, other GSDM family members are also palmitoylated, suggesting that ROS stress and palmitoylation may be a general switch for the activation of this pore-forming family.

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

Healy

David

et al. 2023

Preprint

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“…This suggests that GSDME NT may be subject to additional layers of regulation. Interestingly, GSDMD pore formation may also be further regulated: Efficient GSDMD pore formation relies on ROS generated by the Ragulator-Rag-mTORC1 pathway, which likely mediates the oxidative post-translational modification of GSDMD cysteine 191 10 .…”

Section: Discussionmentioning

confidence: 99%

“…As a result, the plasma membrane becomes permeable to DNA intercalating dyes such as propidium iodide or DRAQ7 and the mature cytokines IL-1β and IL-18 are released 1,3,8 . GSDMD pore formation has recently been found to be enhanced by reactive oxygen species (ROS), which likely mediate oxidative modification of cysteine 192 in murine GsdmdD (corresponding to cysteine 191 in human GSDMD) 9,10 . Eventually, the entire cell ruptures and releases larger cytosolic components into the cellular environment, including tetrameric lactate dehydrogenase (LDH) and pro-inflammatory DAMPs that further promote inflammation.…”

Section: Introductionmentioning

confidence: 99%

Antagonistic nanobodies reveal mechanism of GSDMD pore formation and unexpected therapeutic potential

Schiffelers

Normann

BINDER

et al. 2023

Preprint

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Activation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. Yet, it remained unclear what triggers the conformational changes that allow membrane insertion, as methods to study pore formation in living cells were limited. We raised nanobodies specific for human GSDMD and found two nanobodies that prevent pyroptosis and IL-1β release when expressed in the cytosol of human macrophages. Nanobody binding to GSDMDNTblocked its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNTpartitioned into the plasma membrane, suggesting that pore formation is initiated by insertion of monomers, followed by oligomerization in the target membrane. When GSDMD pore formation was inhibited, cells still underwent caspase-1-dependent apoptosis, likely due to the substantially augmented caspase-1 activity. This hints at a novel layer of regulation of caspase-1 activity by GSDMD pores. Moreover, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the medium prevented cell death by pyroptosis, likely by entering through GSDMD pores and curtailing the assembly of additional pores. GSDMD nanobodies may thus be suitable to treat the ever-growing list of diseases caused by activation of the (non-) canonical inflammasomes.

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Potential Impact of Climate Change-Induced Alterations on Pyroptotic Cell Death in Animal Cells: A Review

Berkel

2024

Mol Biotechnol

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Gasdermin D pore-forming activity is redox-sensitive

Cited by 69 publications

References 41 publications

ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

Antagonistic nanobodies reveal mechanism of GSDMD pore formation and unexpected therapeutic potential

Potential Impact of Climate Change-Induced Alterations on Pyroptotic Cell Death in Animal Cells: A Review

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