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

Abstract

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.

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

“…The same residue has recently been proposed to undergo palmitoylation to facilitate membrane association and full activation 61,62 .…”

An overview of programmed cell death: Apoptosis and pyroptosis—Mechanisms, differences, and significance in organism physiology and pathophysiology

“…It has been shown, that this process is regulated by cellular redox state. Elevated reactive ROS levels may lead to promotion of GSDMD palmitoylation and activation 139 . It can be suggested that the main role of Regulator–Rag in pyroptosis is regulation of ROS and other stimuli that promote ROS production would promote GSDMD oligomerization independently of the Regulator–Rag complex activity 138 …”

“…Elevated reactive ROS levels may lead to promotion of GSDMD palmitoylation and activation. 139 It can be suggested that the main role of Regulator-Rag in pyroptosis is regulation of ROS and other stimuli that promote ROS production would promote GSDMD oligomerization independently of the Regulator-Rag complex activity. 138 Studies have shown that GSDM family members are involved in pyroptotic and apoptotic pathways.…”

Structure and assembly of a bacterial gasdermin pore

“…Besides extrinsic membrane features, bGSDMs appear to broadly utilize intrinsic properties such as palmitoylation to strengthen membrane interactions and induce membrane disorder to lower the energy barrier for pore formation. Building on the identification of bGSDM palmitoylation 15 , two recent studies describe a requirement of enzyme-dependent palmitoylation of human and mouse GSDMD for pore formation in cells 32,33 . Though the palmitoylated cysteine (C191 in humans) is located within the fingertip region instead of on the thumb in bGSDMs, we suggest that our model for palmitoyl membrane insertion may explain why the modification is conserved across the tree of life.…”