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Chemical proteomics identifies a novel off-target protein of small molecule NLRP3 inflammasome inhibitor MCC950

ABSTRACT: Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders. MCC950, a potent inhibitor of the NLR-family inflammasome pyrin domain-containing 3 (NLRP3) protein, has shown encouraging results in animal models for a range of conditions; however, little is known about its off-target effects. We have designed, synthesized and applied a novel photoaffinity alkyne-tagged probe for MCC950 which shows direct engagement with NLRP3 and inhibition of inflammasome activation in macrophages. Affinity-based chemical proteomics in live macrophages identified several potential off-targets, and independent cellular thermal proteomic profiling revealed stabilization of these by MCC950. This is the results of the affinity-based proteomics.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Cassandra Kennedy

LAB HEAD: Edward W Tate

PROVIDER: PXD024913 | Pride | 2021-05-17

REPOSITORIES: Pride

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	CK_Run_A_F1.raw	Raw
	CK_Run_A_F2.raw	Raw
	CK_Run_A_F3.raw	Raw
	CK_Run_A_F4.raw	Raw
	CK_Run_A_F5.raw	Raw

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Similar Datasets

Chemical proteomics identifies a novel off-target protein of small molecule NLRP3 inflammasome inhibitor MCC950

Project description:Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders. MCC950, a potent inhibitor of the NLR-family inflammasome pyrin domain-containing 3 (NLRP3) protein, has shown encouraging results in animal models for a range of conditions; however, little is known about its off-target effects. We have designed, synthesized and applied a novel photoaffinity alkyne-tagged probe for MCC950 which shows direct engagement with NLRP3 and inhibition of inflammasome activation in macrophages. Affinity-based chemical proteomics in live macrophages identified several potential off-targets, and independent cellular thermal proteomic profiling revealed stabilization of these by MCC950. This is the results of the thermal protein profiling.

2021-05-17 | PXD024915 | Pride

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

Project description: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.

2024-04-24 | PXD045862 | Pride

Tyrosine Phosphorylation of NLRP3 by Src Family Protein Tyrosine Kinase Lyn Suppresses NLRP3 Inflammasome Activation

Project description:The NLRP3 inflammasome is a multi-protein complex that triggers the activation of the inflammatory protein caspase-1 and the maturation of the cytokine IL-1 in response to microbes and other danger signals in host cells. Here, we sought a deeper understanding of how the NLRP3 inflammasome is regulated. We found that inflammasome activation induced the Src family kinase Lyn to phosphorylate NLRP3 at Tyr918, and that this phosphorylation of NLRP3 correlated with a subsequent increase in its ubiquitination, which facilitated its proteasome-mediated degradation. NLRP3 tyrosine phosphorylation and ubiquitination was abrogated in Lyn-deficient macrophages, which produced increased amounts of IL-1. Furthermore, mice lacking Lyn were highly susceptible to LPS-induced septic shock in an NLRP3-dependent manner. Our data demonstrate that Lyn-mediated tyrosine phosphorylation of NLRP3 is a prerequisite for its ubiquitination, thus dampening NLRP3 inflammasome activity.

2022-05-19 | PXD028582 | Pride

Lipid droplet-mediated danger signaling balances Trem2+ macrophage induction and NLRP3 inflammasome activation in NASH

Project description:Macrophages are exquisitely capable of sensing danger-associated molecular patterns (DAMPs) and orchestrating inflammatory response during tissue injury. Nonalcoholic steatohepatitis (NASH) represents an advanced stage of metabolic fatty liver disease that increases the risk for cirrhosis and liver cancer. Pathogenic mechanisms of NASH center on hepatocyte injury and the ensuing immune response within the liver microenvironment. However, the nature of DAMPs released by injured hepatocytes and how they shape the liver immune milieu remain largely unknown. Here we show that lipid droplets (LDs) released by injured fatty hepatocytes provide a potent signal that triggers monocyte infiltration and maturation into Trem2+ macrophages, recently described as NASH-associated macrophages (NAMs) or lipid- associated macrophages. LD treatment exacerbated liver injury in mice with diet-induced NASH. We identified Membrane spanning 4-domains a7 (Ms4a7) as a NAM-specific pathogenic factor that was strongly induced in mouse and human NASH. Ms4a7 inactivation ameliorated key aspects of diet-induced NASH pathologies in mice. At the mechanistic level, Ms4a7 physically interacts with NLR family pyrin domain containing 3 (NLRP3) and is required for endosomal NLRP3 inflammasome activation. These findings illustrate a crucial role of Ms4a7 in driving pro-inflammatory signaling in NASH liver. Surprisingly, LD treatments attenuated Ms4a7 expression and NLRP3 inflammasome activation in cultured macrophages. As such, LDs serve as a DAMP signal that balances the induction of Trem2+ macrophages and NLRP3 inflammasome activation in NASH liver

2024-04-26 | GSE230700 | GEO

The inflammasome is a central player in the induction of obesity and insulin resistance

Project description:Inflammation plays a key role in the pathogenesis of obesity. Chronic overfeeding leads to macrophage infiltration in the adipose tissue, resulting in pro-inflammatory cytokine production. Both microbial and endogenous danger signals trigger assembly of the intracellular innate immune sensor Nlrp3 [NLR family, pyrin domain containing 3] resulting in caspase-1 activation and production of pro-inflammatory cytokines interleukin (IL)-1beta and IL-18. Here, we showed that mice deficient in Nlrp3, ASC [apoptosis-associated speck-like protein containing a CARD; a.k.a PYCARD (PYD and CARD domain containing)] and caspase-1 were resistant to the development of high fat diet-induced obesity, which correlated with protection from obesity-induced insulin resistance. Detailed metabolic and molecular phenotyping demonstrated that the inflammasome controls energy expenditure and adipogenic gene expression during chronic overfeeding. These findings reveal a critical function of the inflammasome in obesity and insulin resistance and suggest inhibition of the inflammasome as a potential therapeutic strategy. Keywords: Expression profiling by array Wild-type (WT), ASC-null and Casp1-null mice were subjected to high fat diet feeding for 16 weeks. After the diet intervention period, the animals were killed and epididymal white adipose tissue was removed. Total RNA was isolated and subjected to gene expression profiling.

2011-08-20 | E-GEOD-25205 | biostudies-arrayexpress

FADD is a new protein secreted unconventionally following classical NLRP3 inflammasome activation

Project description:NLRP3 inflammasome assembles in response to stress or danger signals and leads to unconventional secretion of proinflammatory IL-1. FADD is an NLRP3 inflammasome component. Here we found that classical NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion, which required potassium efflux, functional NLRP3 sensor, ASC adaptor and caspase-1 scaffold molecule. FADD is a leaderless protein unconventionally secreted through plasma membrane-derived microvesicles. Blood-derived monocytes from rheumatoid arthritis (RA) patients secreted more FADD following NLRP3 inflammasome activation than those from healthy donors, and we found increased levels of FADD in the sera (ESPOIR cohort) and synovial fluids from RA patients. Levels of synovial FADD correlated with the inflammatory status of the joint. These data reveal that FADD secretion occurs during inflammatory disease in vivo.

2019-02-28 | PXD008168 | Pride

Priming by IKKβ recruits NLRP3 to the trans-Golgi network to enable NEK7-independent inflammasome activation

Project description:The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: The first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here we show that IKKβ, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a lipid enriched on the trans-Golgi network. Intriguingly, NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKβ recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKβ-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a new type of "primed" state into which NLRP3 is brought by IKKβ activity.

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IRF8 regulates transcription of Naips for NLRC4 inflammasome activation

Project description:Inflammasome activation is critical for host defense against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

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The Ragulator complex regulates NLRP3 inflammasome activation through interaction with HDAC6

Project description:The activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but the precise roles of lysosomes are still unclear. Here we show the vital role of the Ragulator complex, a lysosomal protein, in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophage and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed remarkable attenuation of the severity of NLRP3-associated inflammatory diseases, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with histone deacetylase 6 (HDAC6) during NLRP3 inflammasome activation, and this interaction augmented the interaction between the Ragulator complex and NLRP3. Lack of HDAC6 attenuated the interaction between Lamtor1 and NLRP3, resulting in insufficient NLRP3 inflammasome activation. Furthermore, DL-all-rac-α-tocopherol inhibited Lamtor1–HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. Our results provide insight into the role of lysosomes in providing a platform for the activation of NLRP3 inflammasomes by the Ragulator complex.

2022-11-29 | PXD033497 | Pride

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