Understanding the mechanistic basis of palmitoylation in NLRP3 inflammasome activation in disease

Inflammasomes are multi-protein complexes that result in the cleavage of cysteine protease caspase-1 leading to the secretion of a key cytokine IL-1b and the induction of an inflammatory cell death, pyroptosis. The NLRP3 inflammasome, in particular, can be activated by various endogenous danger signals. The overt stimulation of these pathways is detrimental and contributes to the development of inflammatory and metabolic disorders. Moreover, several known mutations in the gene encoding for the sensor protein, NLRP3, are associated to Cryopyrin-Associated Periodic Syndromes (CAPS), a debilitating autoinflammatory condition characterized by recurrent episodes of fever, rash, joint pain, and other associated symptoms. It is known that the proteins forming the inflammasome are localised in different cellular compartments and must come together at the endosomal/lysosomal compartment to achieve full assembly and activation. However, we have little understanding of the mechanisms that enable this localisation. Moreover, how inflammasome-dependent inflammation is ultimately resolved remains unknown, but holds therapeutic potential for a range of diseases. In this application, we aim to explore novel concepts that result in inflammasome localisation and activation, how the inflammasome is subsequently uncoupled, and the role of inflammasome in NLRP3-dependent autoinflammation. We propose that the activation of the inflammasome is centrally dependent on a lipid modification of NLRP3 – specifically, palmitoylation, the conjugation of palmitate to target proteins. We propose that this post-translational modification allows NLRP3 to recruit to the endolysosomal compartment for activation. We posit that modulation of palmitoylation is what governs inflammasome activity, with NLRP3 failing to lose the palmitate modification in disease. By examining the dynamics of NLRP3 palmitoylation in relation to its spatial localisation, we seek to unravel new mechanisms that drive inflammasome activation. Our aim is to uncover these fundamental mechanisms and extend them to understand how inflammasome regulation is dysregulated in disease. The NLRP3 inflammasome is established to instigate and advance a range of disorders, including atherosclerosis, diabetes, Alzheimer’s, and obesity. In deciphering these mechanisms, we will probe the role of a specific endolysosomal enzyme (depalmitoylase) that we propose removes the palmitate modification from NLRP3 after inflammasome activation. Failure to remove palmitate may result in prolonged NLRP3 activation, risking sustained inflammation. We believe we have a unique opportunity to examine this key mechanism and generate insights that will be relevant to a spectrum of NLRP3-related disorders. Our objectives are: Investigate the significance of palmitoylation in NLRP3 gain-of-function mutants. We will examine whether the NLRP3 disease variants undergo a) palmitoylation and, importantly, b) depalmitoylation. We will also examine the nature of upstream stimuli driving the lipid modification in CAPS. Dissect the role of depalmitoylase in NLRP3 inflammasome activation. Here, we will study the expression, regulation, and involvement of a key depalmitoylase in inflammasome activation. Determine the physiological role of palmitoylation and PPT1 in NLRP3 autoinflammation. We will employ experimental models including those expressing NLRP3 gain-of-function mutants to determine the physiological function of the two activities.