Spatiotemporal proteomic profiling of cellular responses to NLRP3 agonists

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 3 (NLRP3) is an innate immune sensor that forms an inflammasome in response to various cellular stressors. Gain-of-function mutations in NLRP3 cause autoinflammatory diseases and NLRP3 signalling itself exacerbates the pathogenesis of many other human diseases. Despite considerable therapeutic interest, the primary drivers of NLRP3 activation remain controversial due to the diverse array of signals that are integrated through NLRP3. Here, we mapped subcellular proteome changes to lysosomes, mitochondrion, EEA1-positive endosomes, and Golgi caused by the NLRP3 inflammasome agonists nigericin and CL097. We identified several common disruptions to retrograde trafficking pathways, including COPI and Shiga toxin-related transport, in line with recent studies. We further characterized mouse NLRP3 trafficking throughout its activation using temporal proximity proteomics, which supports a recent model of NLRP3 recruitment to endosomes during inflammasome activation. Collectively, these findings provide additional granularity to our understanding of the molecular events driving NLRP3 activation and serve as a valuable resource for cell biological research. We have made our proteomics data accessible through an open-access Shiny browser to facilitate future research within the community, available at: https://harperlab.connect.hms.harvard.edu/inflame/. We will display anonymous peer review for this manuscript on pubpub.org (https://harperlab.pubpub.org/pub/nlrp3/) rather than a traditional journal. Moreover, we invite community feedback on the pubpub version of this manuscript, and we will address criticisms accordingly.


Figure 3 supporting figures
Background control IPs separate well from MitoIPs.(E) Violin plots depicting the log 2 fold change (FC) values for the indicated subcellular compartment in aggregate for the untreated MitoIP (HEK293T M ) compared to the background control (HEK293T-CTR).Mitochondrial proteins are enriched over all organellar groups for each condition, validating the approach.(F) PCA colored as in (C) with background samples excluded.Replicates of a given condition cluster well.(G) Violin plots depicting the log 2 FC values for the indicated subcellular compartment and the given treatment condition (nigericin or CL097) compared to untreated (MitoTag) cells.Proteins were first filtered for significant mitochondrial localization (any 293T M /293T Control : q < 0.05, Log 2 FC > 0.5).
(H) Violin plots depicting the log 2 FC values for the indicated mitochondrial annotation and the given treatment condition compared to untreated (MitoTag) cells.Proteins were first filtered for significant mitochondrial localization (any 293T M /293T Control : q < 0.05, Log 2 FC > 0.5).
To Figure 3: https://harperlab.pubpub.org/pub/nlrp3#nyjwc0zexqnEndosomal, lysosomal, and plasma membrane proteins (groups that likely traffic through endosomes) are enriched over all organellar annotations for each condition (indicated by arrows), validating the approach.(E) Principal component analysis (PCA) colored as in (C) with all channels included.
Replicates of a given condition correlate well.(F) Violin plots depicting the log 2 FC values for the indicated organelle annotation and the given treatment condition compared to untreated (EndoTag) cells.Proteins were first filtered for significant endosomal localization (any 293T EG /293T: q < 0.05, Log 2 FC > 0.5).

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Figure 5 -
Figure 5-Supporting Figure 3. Protein group analysis for the GolgiIP experiment.(A-B) GO-term analysis for proteins that significantly increased in the indicated GolgiIP versus untreated cells (Log 2 FC > 0.5, q < 0.05).(C-D) Analysis of the top 20 Bioplex protein interactomes (>3 proteins) positively enriched in the indicated experiment.V-ATPase interactors are in bold and italicized.(E-F) GO-term analysis for proteins that significantly decreased in the indicated LysoIP versus untreated cells (Log 2 FC < -0.5, q < 0.05).(G-H)Analysis of the top 20 Bioplex protein interactomes (>3 proteins) depleted in the indicated experiment.COPI subunits are in bold, and their COPI-associated proteins are in bold and italicized.To Figure5: https://harperlab.pubpub.org/pub/nlrp3#nkrmiksruzv

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Figure 6 supporting figures