| Summary: | Inflammasomes are multi-protein complexes that serve as critical sensors to fend off pathogens and resolve aberrant cellular physiology. The NOD-, LRR-, and Pyrin domain-containing 3 (NLRP3) inflammasome can detect a wide range of stimuli, including pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and homeostasis-altering molecular processes (HAMPs). It drives the production of pro-inflammatory cytokine interleukin-1β (IL-1β) through the caspase-1 dependent signalling pathway. Despite extensive studies, no consensus model for the activation mechanism exists. Thus, systematic interrogation of NLRP3 inflammasome activation is highly desirable. Emerging mass spectrometry (MS) methodologies and proteomics have enabled the comprehensive mapping of intracellular protein networks. In this thesis, multiple MS-based strategies have been utilised to examine the global perturbation during NLRP3 inflammasome priming and activation. Using multiplexed TMT-based proteomics and di-Gly ubiquitomics, we presented a high-resolution map of proteome and ubiquitome profiles in THP-1 cells in response to NLRP3 activators. Through the combination of co-fractionation mass spectrometry (CF-MS) and machine learning algorithms, we elucidated the spatial and subcellular proteomic changes during the NLRP3 inflammasome activation process. We observe substantial relocalisation of the complex upon stimulation. Furthermore, utilising the APEX2 proximity labelling (PL)-MS approach, we spatiotemporally characterized the “proximity proteome (also known as proximitome)” of the NLRP3 inflammasome. Combined with RNAi screening, two NLRP3-interacting proteins, USP10 and UCHL1, were identified and validated as essential regulators for NLRP3 activation downstream of potassium efflux. Collectively, the MS-based approaches, including multiplexed proteomics, di-Gly ubiquitomics, CF-MS, and APEX2 PL-MS, enable a comprehensive understanding of the NLRP3 inflammasome activation process. These new insights into the underlying mechanism provide potential novel therapeutic targets, which may ultimately lead to treatments for NLRP3-associated diseases.
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