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Proximity proteomics reveals UCH-L1 as an essentialregulator of NLRP3-mediated IL-1β production in humanmacrophages and microglia

ABSTRACT: Activation of the NACHT, LRR family pyrin domain containing 3 (NLRP3) inflammasome complex is an essential innate immune signalling mechanism. To reveal how NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labelling, affinity purification and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. We discovered that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3, and downregulation of UCH-L1 decreases pro-IL-1β levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerisation, leading to altered IL-1β cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1β production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Kidney

DISEASE(S): Inflammation

SUBMITTER: Zhu Liang

LAB HEAD: Benedikt Kessler

PROVIDER: PXD045862 | Pride | 2024-04-24

REPOSITORIES: Pride

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			Action	DRS
	FL1270_MSQ2015_20201006_ZLiang_10_Nig-30_1.raw	Raw
	FL1270_MSQ2015_20201006_ZLiang_11_Nig-60_1.raw	Raw
	FL1270_MSQ2015_20201006_ZLiang_1_beads_1.raw	Raw
	FL1270_MSQ2015_20201006_ZLiang_2_beads_2.raw	Raw
	FL1270_MSQ2015_20201006_ZLiang_3_beads_3.raw	Raw

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Project description:BACKGROUND: Cryopyrin-Associated Periodic Syndrome (CAPS) is an autoinflammatory condition consequence of monoallelic variants in the NLRP3 gene that exacerbate IL-1β production. These variants are gain-of-function, but the exact regulatory mechanism of the NLRP3 CAPS-inflammasome is not well yet understood. It is considered as a hypersensitive inflammasome triggered by cell priming, but patients with CAPS and animal disease models present inflammatory flares in the absence of external triggers.OBJECTIVES: To study the regulation and activation of the NLRP3 inflammasome in CAPS.METHODS: CAPS derived blood samples and genetically modified macrophages expressing different NLRP3 CAPS-associated variants were used to assess NLRP3 function dissociated from cell priming and cell metabolism.RESULTS: We found that CAPS-associated variants constitutively result in active NLRP3 inflammasomes, that induce a basal cleavage of gasdermin D, IL-18 release and pyroptosis. The constitutive active NLRP3 inflammasome was blocked by MCC950 and was dependent on NLRP3 expression level, being further regulated by deubiquitination. We also showed that activation of NF-κB with lipopolysaccharide or other endogenous host-derived molecules (palmitate, S100A9 or IL-6) further modulated the activation of the NLRP3 CAPS inflammasome and expanded the repertoire of molecules secreted from CAPS macrophages to IL-1β, IL-1α, HMGB1, cystatin B and the P2X7 receptor, identifying novel proteins involved in CAPS pathogenesis. NLRP3 inflammasomes with CAPS associated variants affected the immunometabolism of the myeloid compartment and impaired glycolysis.CONCLUSIONS: These findings demonstrate that NLRP3 CAPS-associated variants form a constitutive active inflammasome that induce basal pyroptosis and IL-18 release without cell priming, suggesting a priming-independent mechanism for the initiation of CAPS flares characterized with a profound affection in the immunometabolism.

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Proximity proteomics reveals UCH-L1 as an essentialregulator of NLRP3-mediated IL-1β production in humanmacrophages and microglia

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