Temporal dissection of NLRP3 protein-protein interactions in inflammasome regulation

The NLRP3 inflammasome is a central signalling hub of the innate immune system which mediates inflammation through pro-inflammatory cytokine release and inflammatory cell death (pyroptosis). Dysregulated NLRP3 inflammasome signalling underlies many human diseases. The NLRP3 pathway undergoes a complex two-step programme of priming and activation modulated by dynamic protein-protein interactions (PPIs) and post-translational modifications (PTMs). However, the contextual and temporal dynamics of these mechanisms, and how they act to regulate NLRP3, are not completely understood. Previous studies have shown that temporal dynamics of PPI networks and corresponding PTMs can be assessed by affinity purification coupled to quantitative mass spectrometry (AP-MS) (1,2) and correlations between protein complex formation and post-translational modifications can be assessed, indicative of functional relationships (2). This project aims to build a comprehensive temporal map of PPI/PTM dynamics in NLRP3 inflammasome activation identifying novel regulatory mechanisms and their functional consequences.

To derive a time-resolved quantitative map of reorganization of protein-protein interactions and corresponding PTMs we affinity purified NLRP3 from cells across a time course of priming and activation stimuli. We have established and validated protocols for AP-MS of endogenously expressed twin-strep tagged NLRP3 following stimulation of 5x106 THP-1 monocytes cells. The enhanced sensitivity and speed of diaPASEF (3) enabled quantitative analysis over many conditions and time-points including 150+ samples from 38 distinct experimental conditions.

Time points were selected to focus on poorly understood early events (i.e. within 15 minutes of stimulation) in both priming and activating stimulation conditions and examine NLRP3 interactome immediately following stimulus detection. High confidence interactors have been identified for each stimulation timepoint via filtering against untagged (wild type) NLRP3 background controls. These interactions have been validated by the presence of known NLRP3 interacting proteins, and include putative novel interacting partners. Ongoing analysis is examining the relationships of detected PPIs and PTMs of NLRP3 and will attempt to define their functional significance for NLRP3 priming and activation and its phenotypic consequences.

We show that AP-MS using diaPASEF enables in depth quantitative analysis of NLRP3 protein interaction dynamics across finely resolved time points and diverse stimulation conditions providing new insights into this complex and clinically important innate immune signalling pathway.

References:
1. Collins BC, et al. Nature Methods. 2013;10(12):1246–53.
2. Zheng Y, et al. Nature. 2013;499(7457):166–71.
3. Meier F, et al. Nature Methods. 2020;17(12):1229–36.