RESUMO
Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1ß production. Cathepsin Z has been associated with the development of inflammation and IL-1ß production; however, the mechanism of how cathepsin Z leads to IL-1ß production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using WT mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using WT and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. The cells were activated with silica, and IL-1ß release was determined using enzyme-linked immunosorbent assay or IL-1ß bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1ß expression in response to silica. The secreted cathepsin Z functions nonproteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1ß through integrin α5 without affecting the transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.
Assuntos
Catepsina Z , Inflamassomos , Animais , Catepsina Z/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Integrina alfa5/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Dióxido de Silício/farmacologia , Silicose/metabolismoRESUMO
In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1ß release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1ß release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein-ligand binding that might explain the activity of the compounds.
Assuntos
Imidazóis , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células THP-1RESUMO
The prototypical model for NOD-like receptor (NLR) inflammasome assembly includes nucleotide-dependent activation of the NLR downstream of pathogen- or danger-associated molecular pattern (PAMP or DAMP) recognition, followed by nucleation of hetero-oligomeric platforms that lie upstream of inflammatory responses associated with innate immunity. As members of the STAND ATPases, the NLRs are generally thought to share a similar model of ATP-dependent activation and effect. However, recent observations have challenged this paradigm to reveal novel and complex biochemical processes to discern NLRs from other STAND proteins. In this review, we highlight past findings that identify the regulatory importance of conserved ATP-binding and hydrolysis motifs within the nucleotide-binding NACHT domain of NLRs and explore recent breakthroughs that generate connections between NLR protein structure and function. Indeed, newly deposited NLR structures for NLRC4 and NLRP3 have provided unique perspectives on the ATP-dependency of inflammasome activation. Novel molecular dynamic simulations of NLRP3 examined the active site of ADP- and ATP-bound models. The findings support distinctions in nucleotide-binding domain topology with occupancy of ATP or ADP that are in turn disseminated on to the global protein structure. Ultimately, studies continue to reveal how the ATP-binding and hydrolysis properties of NACHT domains in different NLRs integrate with signaling modules and binding partners to control innate immune responses at the molecular level.
Assuntos
Trifosfato de Adenosina/metabolismo , Inflamassomos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Hidrólise , Simulação de Dinâmica Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
The pregnane X receptor (PXR) is a ligand-activated nuclear receptor that acts as a xenobiotic sensor, responding to compounds of foreign origin, including pharmaceutical compounds, environmental contaminants, and natural products, to induce transcriptional events that regulate drug detoxification and efflux pathways. As such, the PXR is thought to play a key role in protecting the host from xenobiotic exposure. More recently, the PXR has been reported to regulate the expression of innate immune receptors in the intestine and modulate inflammasome activation in the vasculature. In the current study, we report that activation of the PXR in primed macrophages triggers caspase-1 activation and interleukin-1ß release. Mechanistically, we show that this response is nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3-dependent and is driven by the rapid efflux of ATP and P2X purinoceptor 7 activation following PXR stimulation, an event that involves pannexin-1 gating, and is sensitive to inhibition of Src-family kinases. Our findings identify a mechanism whereby the PXR drives innate immune signaling, providing a potential link between xenobiotic exposure and the induction of innate inflammatory responses.
Assuntos
Trifosfato de Adenosina/metabolismo , Inflamassomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor de Pregnano X/metabolismo , Animais , Caspase 1/metabolismo , Linhagem Celular Tumoral , Conexinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Interleucina-1beta/metabolismo , Cinética , Ligantes , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptor de Pregnano X/agonistas , Receptores Purinérgicos P2X7/metabolismo , Quinases da Família src/metabolismoRESUMO
The pyrin domain containing Nod-like receptors (NLRPs) are a family of pattern recognition receptors known to regulate an array of immune signaling pathways. Emergent studies demonstrate the potential for regulatory control of inflammasome assembly by phosphorylation, notably NLRP3. Over a dozen phosphorylation sites have been identified for NLRP3 with many more suggested by phosphoproteomic studies of the NLRP family. Well characterized NLRP3 phosphorylation events include Ser198 by c-Jun terminal kinase (JNK), Ser295 by protein kinase D (PKD) and/or protein kinase A (PKA), and Tyr861 by an unknown kinase but is dephosphorylated by protein tyrosine phosphatase non-receptor 22 (PTPN22). Since the PKA- and PKD-dependent phosphorylation of NLRP3 at Ser295 is best characterized, we provide detailed review of this aspect of NLRP3 regulation. Phosphorylation of Ser295 can attenuate ATPase activity as compared to its dephosphorylated counterpart, and this event is likely unique to NLRP3. In silico modeling of NLRP3 is useful in predicting how Ser295 phosphorylation might impact upon the structural topology of the ATP-binding domain to influence catalytic activity. It is important to gain as complete understanding as possible of the complex phosphorylation-mediated mechanisms of regulation for NLRP3 in part because of its involvement in many pathological processes.
Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , FosforilaçãoRESUMO
The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.
Assuntos
Trifosfato de Adenosina/metabolismo , Inflamassomos/metabolismo , Proteínas NLR/metabolismo , Células HEK293 , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-ß-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation.
Assuntos
Neoplasias da Mama , Inibidores de Histona Desacetilases , Feminino , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Cromatina , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Vorinostat/farmacologiaRESUMO
During invasion, Entamoeba histolytica (Eh) encounter macrophages and activate them to elicit tissue damaging pro-inflammatory responses. When Eh binds macrophages via the Gal-lectin, surface EhCP-A5 RGD sequence ligates α5ß1 integrin to activate caspase-1 in a complex known as the NLRP3 inflammasome. In this study, we investigated Eh requirements underlying macrophage caspase-4 and -1 activation and the role caspase-4 and gasdermin D (GSDMD) play in augmenting pro-inflammatory cytokine responses. Caspase-4 activation was similar to caspase-1 requiring live Eh attachment via the Gal-lectin and EhCP-A5. However, unlike caspase-1, caspase-4 activation was independent of ASC and NLRP3. Using CRISPR/Cas9 gene editing of caspase-4 and -1 and GSDMD, we determined that caspase-1 and bioactive IL-1ß release was highly dependent on caspase-4 activation and cleavage of GSDMD in response to Eh. Formaldehyde cross-linking to stabilize protein-protein interactions in transfected COS-7 cells stimulated with Eh revealed that caspase-4 specifically interacted with caspase-1 in a protein complex that enhanced the cleavage of caspase-1 CARD domains to augment IL-1ß release. Activated caspase-4 and -1 cleaved GSDMD liberating the N-terminal p30 pore-forming fragment that caused the secretion of IL-1ß. These findings reveal a novel role for caspase-4 as a sensor molecule to amplify pro-inflammatory responses when macrophage encounters Eh.
Assuntos
Caspases Iniciadoras/metabolismo , Entamoeba histolytica/fisiologia , Entamebíase/imunologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Proteínas de Neoplasias/metabolismo , Animais , Células COS , Caspase 1/metabolismo , Caspases Iniciadoras/genética , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Integrina alfa5beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Ligação a Fosfato , Células THP-1RESUMO
The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1ß secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1ß production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.
Assuntos
Caspases Iniciadoras/metabolismo , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Toxina Shiga/farmacologia , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Proteínas de Ligação a Fosfato , Piroptose/efeitos dos fármacosRESUMO
Radiographic contrast agents cause acute kidney injury (AKI), yet the underlying pathogenesis is poorly understood. Nod-like receptor pyrin containing 3-deficient (Nlrp3-deficient) mice displayed reduced epithelial cell injury and inflammation in the kidney in a model of contrast-induced AKI (CI-AKI). Unexpectedly, contrast agents directly induced tubular epithelial cell death in vitro that was not dependent on Nlrp3. Rather, contrast agents activated the canonical Nlrp3 inflammasome in macrophages. Intravital microscopy revealed diatrizoate (DTA) uptake within minutes in perivascular CX3CR1+ resident phagocytes in the kidney. Following rapid filtration into the tubular luminal space, DTA was reabsorbed and concentrated in tubular epithelial cells via the brush border enzyme dipeptidase-1 in volume-depleted but not euvolemic mice. LysM-GFP+ macrophages recruited to the kidney interstitial space ingested contrast material transported from the urine via direct interactions with tubules. CI-AKI was dependent on resident renal phagocytes, IL-1, leukocyte recruitment, and dipeptidase-1. Levels of the inflammasome-related urinary biomarkers IL-18 and caspase-1 were increased immediately following contrast administration in patients undergoing coronary angiography, consistent with the acute renal effects observed in mice. Taken together, these data show that CI-AKI is a multistep process that involves immune surveillance by resident and infiltrating renal phagocytes, Nlrp3-dependent inflammation, and the tubular reabsorption of contrast via dipeptidase-1.