The endogenous context-dependent interactome of the human inflammasome sensor NLRP3

We validated a THP-1 monocyte-like cell line endogenously expressing Twin-Strep tagged (TST) NLRP3, which retain functional NLRP3 inflammasome priming and activation. Using affinity purification coupled to quantitative mass spectrometry, we performed purification and profiling of NLRP3 associated protein complexes across 26 time points and stimulation conditions. Strict statistical filtering of this data revealed several previously observed NLRP3 interacting proteins as well as a large set of putative novel NLRP3 interacting partners.

Functional enrichment analysis of these interactors revealed their roles in several NLRP3 activation associated processes such as protein kinase activity, regulation of cellular trafficking, and mitochondrial electron transport chain function. Among these novel interacting proteins were several protein kinases not previously shown to interact with NLRP3. Given that phosphorylation is a key regulatory process in NLRP3 activation, these were prioritised for functional characterisation.

Preliminary experiments indicated that inhibition of the kinase activity of MAPK family members ASK1 and JNK2/JNK3 inhibited NLRP3 inflammasome activation. Altogether, this work represents a significant step towards understanding the complex regulatory mechanisms associated with the NLRP3 inflammasome and will enable further investigation within the NLRP3 research community.<br><br><i>Thesis is embargoed until 31 December 2026.</i>