NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi.

The molecular mechanisms leading to saliva secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren’s syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of salivary gland immune cell infiltration and glandular hypofunction. SS-like disease was induced by treatment with DMXAA, a small molecule agonist of murine STING. We have previously shown that the extent of salivary secretion is correlated with the magnitude of intracellular Ca²⁺ signals (Takano et al., 2021). Contrary to our expectations, despite a significant reduction in fluid secretion, neural stimulation resulted in enhanced Ca²⁺ signals with altered spatiotemporal characteristics in vivo. Muscarinic stimulation resulted in reduced activation of the Ca²⁺-activated Cl^- channel, TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca²⁺. Super-resolution microscopy revealed a disruption in the colocalization of Inositol 1,4,5-trisphosphate receptor Ca²⁺ release channels with TMEM16a, and channel activation was reduced when intracellular Ca²⁺ buffering was increased. These data indicate altered local peripheral coupling between the channels. Appropriate Ca²⁺ signaling is also pivotal for mitochondrial morphology and bioenergetics. Disrupted mitochondrial morphology and reduced oxygen consumption rate were observed in DMXAA-treated animals. In summary, early in SS disease, dysregulated Ca²⁺ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction.