A group of researchers led by Prof Benedikt Kessler, Professor of Biochemistry and Mass Spectrometry, Dr Zhu Liang and Dr Andreas Damianou employed APEX2 proximity labelling coupled with mass spectrometry to map the NLRP3 proximal proteome (entire complement of proteins expressed) under resting and activated conditions. Activation of the NLRP3 inflammasome complex is an essential innate immune signalling mechanism. APEX2 labelling allows the identification and isolation of endogenous proteins.
Using complementary techniques, such as APEX2 PL-MS, AP-MS, and small interfering (siRNA) screening, they traced the time-resolved protein-protein interaction networks local to the NLRP3 inflammasome upon stimulation. This approach offers a resource-rich map for further investigations of the molecular intricacies of NLRP3 inflammasome activation and reveals that ubiquitin C-terminal hydrolase 1 (UCH-L1) is involved in regulating IL-1β production.
Further experiments in both a reconstituted system and macrophages demonstrate that UCH-L1 interacts with the NACHT domain of NLRP3 and thereby interferes with ASC and NLRP3 assembly. Furthermore, UCH-L1 knockdown or chemical inhibition interferes with IL-1β production, particularly in microglia cells that exhibited elevated UCH-L1 expression as compared to peripheral monocytes or macrophages. The authors propose that UCH-L1 may play a "gatekeeper" role in NLRP3 inflammasome assembly, potentially through modulating ubiquitination of NLRP3.
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