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1.
Nat Immunol ; 21(8): 880-891, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32541830

RESUMO

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-caspase-4 pathway in antibacterial defense.


Assuntos
Caspases Iniciadoras/imunologia , Proteínas de Ligação ao GTP/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Inflamassomos/imunologia , Transdução de Sinais/imunologia , Animais , Bactérias Gram-Negativas/imunologia , Células HeLa , Humanos , Lipopolissacarídeos/imunologia , Camundongos , Piroptose/imunologia
2.
EMBO J ; 37(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29459437

RESUMO

Pathogenic and commensal Gram-negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase-11 non-canonical inflammasome. Here, we show that OMV-induced inflammasome activation requires TLR4-TRIF signaling, the production of type I interferons, and the action of guanylate-binding proteins (GBPs), both in macrophages and in vivo Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram-negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell-autonomous immunity and innate immunity.


Assuntos
Bactérias/imunologia , Membrana Celular/imunologia , Proteínas de Ligação ao GTP/imunologia , Inflamassomos/imunologia , Lipopolissacarídeos/imunologia , Animais , Proteínas de Ligação ao GTP/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout
3.
EMBO Rep ; 21(11): e50829, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33124769

RESUMO

Inflammatory caspase-11 (rodent) and caspases-4/5 (humans) detect the Gram-negative bacterial component LPS within the host cell cytosol, promoting activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1-related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but non-deleterious inflammatory responses. Here, we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively counteract Gram-negative bacteria-induced non-canonical inflammasome activation, both in cultured macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lowers caspase-11-mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)-dependent and GBP-independent routes for caspase-11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine-tune bacteria-activated non-canonical inflammasome responses and shed light on the understanding of the immune pathways they control.


Assuntos
Caspases , Lipopolissacarídeos , Família da Proteína 8 Relacionada à Autofagia , Caspases/genética , Caspases Iniciadoras , Bactérias Gram-Negativas , Inflamassomos/genética , Macrófagos
4.
EMBO Rep ; 20(9): e48235, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31353801

RESUMO

Caspase-4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non-canonical inflammasome, which drives inflammatory responses during Gram-negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome-wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS-mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase-4 levels in human monocytes and iPSC-derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non-canonical inflammasome. Upon IFN-γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase-4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram-negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non-canonical inflammasome.


Assuntos
Caspases Iniciadoras/metabolismo , Fator Regulador 2 de Interferon/metabolismo , Caspases Iniciadoras/genética , Morte Celular/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 2 de Interferon/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Células U937
5.
J Biol Chem ; 293(32): 12563-12575, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29929983

RESUMO

The inflammasome is a major component of the innate immune system, and its main function is to activate caspase-1, a cysteine protease that promotes inflammation by inducing interleukin-1ß (IL-1ß) maturation and release into the extracellular milieu. To prevent uncontrolled inflammation, this complex is highly regulated. When it is assembled, the inflammasome is insoluble, which has long precluded the analysis of its interactions with other proteins. Here we used the proximity-dependent biotinylation assay (BioID) to identify proteins associated with caspase-1 during inflammasome activation. Using the BioID in a cell-free system in which the inflammasome had been activated, we found that a caspase-1-biotin ligase fusion protein selectively labeled 111 candidates, including the p62/sequestosome-1 protein (p62). Using co-immunoprecipitation experiments, we demonstrated that p62 interacts with caspase-1. This interaction promoted caspase-1-mediated cleavage of p62 at Asp-329. Mechanistic and functional analyses revealed that caspase-1-mediated cleavage of p62 leads to loss of its interaction with the autophagosomal protein microtubule-associated protein 1 light chain 3 ß (LC3B). Strikingly, overexpression of a p62 N-terminal fragment generated upon caspase-1 cleavage decreased IL-1ß release, whereas overexpression of p62's C-terminal portion enhanced IL-1ß release, by regulating pro-IL1ß levels. Overall, the overexpression of both fragments together decreased IL-1ß release. Taken together, our results indicate that caspase-1-mediated p62 cleavage plays a complex role in balancing caspase-1-induced inflammation.


Assuntos
Apoptose , Caspase 1/metabolismo , Inflamassomos , Interleucina-1beta/metabolismo , Proteína Sequestossoma-1/metabolismo , Coloração e Rotulagem/métodos , Animais , Bioensaio , Biotinilação , Caspase 1/genética , Células HEK293 , Humanos , Camundongos , Proteína Sequestossoma-1/genética
6.
PLoS Pathog ; 13(10): e1006630, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28968459

RESUMO

Guanylate binding proteins (GBPs) are interferon-inducible proteins involved in the cell-intrinsic immunity against numerous intracellular pathogens. The molecular mechanisms underlying the potent antibacterial activity of GBPs are still unclear. GBPs have been functionally linked to the NLRP3, the AIM2 and the caspase-11 inflammasomes. Two opposing models are currently proposed to explain the GBPs-inflammasome link: i) GBPs would target intracellular bacteria or bacteria-containing vacuoles to increase cytosolic PAMPs release ii) GBPs would directly facilitate inflammasome complex assembly. Using Francisella novicida infection, we investigated the functional interactions between GBPs and the inflammasome. GBPs, induced in a type I IFN-dependent manner, are required for the F. novicida-mediated AIM2-inflammasome pathway. Here, we demonstrate that GBPs action is not restricted to the AIM2 inflammasome, but controls in a hierarchical manner the activation of different inflammasomes complexes and apoptotic caspases. IFN-γ induces a quantitative switch in GBPs levels and redirects pyroptotic and apoptotic pathways under the control of GBPs. Furthermore, upon IFN-γ priming, F. novicida-infected macrophages restrict cytosolic bacterial replication in a GBP-dependent and inflammasome-independent manner. Finally, in a mouse model of tularemia, we demonstrate that the inflammasome and the GBPs are two key immune pathways functioning largely independently to control F. novicida infection. Altogether, our results indicate that GBPs are the master effectors of IFN-γ-mediated responses against F. novicida to control antibacterial immune responses in inflammasome-dependent and independent manners.


Assuntos
Francisella tularensis/imunologia , Proteínas de Ligação ao GTP/imunologia , Inflamassomos/imunologia , Interferon gama/imunologia , Tularemia/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Francisella , Técnicas de Silenciamento de Genes , Infecções por Bactérias Gram-Negativas/imunologia , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
7.
Curr Top Microbiol Immunol ; 397: 229-56, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27460813

RESUMO

Francisella tularensis is a facultative intracellular bacterium causing tularemia, a zoonotic disease. Francisella replicates in the macrophage cytosol and eventually triggers cytosolic immune responses. In murine macrophages, Francisella novicida and Francisella tularensis live vaccine strain lyse in the host cytosol and activate the cytosolic DNA receptor Aim2. Here, we review the mechanisms leading or contributing to Aim2 inflammasome activation, including the role of TLRs and of IFN signaling and the implication of the guanylate-binding proteins 2 and 5 in triggering cytosolic bacteriolysis. Furthermore, we present how this cytosolic Gram-negative bacterium escapes recognition by caspase-11 but can trigger a non-canonical caspase-8 inflammasome. In addition, we highlight the differences in inflammasome activation in murine and human cells with pyrin, NLRP3, and AIM2 involved in sensing Francisella in human phagocytes. From a bacterial prospective, we describe the hiding strategy of Francisella to escape recognition by innate sensors and to resist to bacteriolysis in the host cytosol. Finally, we discuss the inability of the inflammasome sensors to detect F. tularensis subspecies tularensis strains, making them highly pathogenic stealth microbes.


Assuntos
Citosol/imunologia , Francisella tularensis/imunologia , Inflamassomos/imunologia , Tularemia/imunologia , Animais , Citosol/microbiologia , Francisella tularensis/genética , Francisella tularensis/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Inflamassomos/genética , Tularemia/microbiologia
8.
Respir Res ; 17(1): 129, 2016 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-27751187

RESUMO

BACKGROUND: In acutely injured lungs, massively recruited polymorphonuclear neutrophils (PMNs) secrete abnormally neutrophil elastase (NE). Active NE creates a localized proteolytic environment where various host molecules are degraded leading to impairment of tissue homeostasis. Among the hallmarks of neutrophil-rich pathologies is a disrupted epithelium characterized by the loss of cell-cell adhesion and integrity. Epithelial-cadherin (E-cad) represents one of the most important intercellular junction proteins. E-cad exhibits various functions including its role in maintenance of tissue integrity. While much interest has focused on the expression and role of E-cad in different physio- and physiopathological states, proteolytic degradation of this structural molecule and ensuing potential consequences on host lung tissue injury are not completely understood. METHODS: NE capacity to cleave E-cad was determined in cell-free and lung epithelial cell culture systems. The impact of such cleavage on epithelial monolayer integrity was then investigated. Using mice deficient in NE in a clinically relevant experimental model of acute pneumonia, we examined whether degraded E-cad is associated with lung inflammation and injury and whether NE contributes to E-cad cleavage. Finally, we checked for the presence of both degraded E-cad and NE in bronchoalveolar lavage samples obtained from patients with exacerbated COPD, a clinical manifestation characterised by a neutrophilic inflammatory response. RESULTS: We show that NE is capable of degrading E-cad in vitro and in cultured cells. NE-mediated degradation of E-cad was accompanied with loss of epithelial monolayer integrity. Our in vivo findings provide evidence that NE contributes to E-cad cleavage that is concomitant with lung inflammation and injury. Importantly, we observed that the presence of degraded E-cad coincided with the detection of NE in diseased human lungs. CONCLUSIONS: Active NE has the capacity to cleave E-cad and interfere with its cell-cell adhesion function. These data suggest a mechanism by which unchecked NE participates potentially to the pathogenesis of neutrophil-rich lung inflammatory and tissue-destructive diseases.


Assuntos
Lesão Pulmonar Aguda/enzimologia , Caderinas/metabolismo , Células Epiteliais/enzimologia , Elastase de Leucócito/metabolismo , Pulmão/enzimologia , Neutrófilos/enzimologia , Pneumonia Bacteriana/enzimologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Antígenos CD , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/patologia , Elastase de Leucócito/deficiência , Elastase de Leucócito/genética , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/patologia , Proteólise
9.
Biochim Biophys Acta ; 1833(8): 1936-46, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23602969

RESUMO

The differentiation of human peripheral blood monocytes into macrophages can be reproduced ex vivo by culturing the cells in the presence of colony-stimulating factor 1 (CSF1). Using microarray profiling to explore the role of microRNAs (miRNAs), we identified a dramatic decrease in the expression of the hematopoietic specific miR-142-3p. Up- and down-regulation of this miRNA in primary human monocytes altered CSF1-induced differentiation of monocytes, as demonstrated by changes in the expression of the cell surface markers CD16 and CD163. One of the genes whose expression is repressed by miR-142-3p encodes the transcription factor Early Growth Response 2 (Egr2). In turn, Egr2 associated with its co-repressor NGFI-A (Nerve Growth Factor-Induced gene-A) binding protein 2 (NAB2) binds to the pre-miR-142-3p promoter to negatively regulate its expression. Interestingly, the expression of miR-142-3p is abnormally low in monocytes from patients with the most proliferative forms of chronic myelomonocytic leukemia (CMML), and miR-142-3p re-expression in CMML dysplastic monocytes can improve their differentiation potential. Altogether, miR-142-3p which functions in a molecular circuitry with Egr2 is an actor of CSF1-induced differentiation of human monocytes whose expression could be altered in CMML.


Assuntos
Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , MicroRNAs/genética , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Células K562 , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/patologia , Macrófagos/citologia , Macrófagos/metabolismo , MicroRNAs/biossíntese , MicroRNAs/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Regulação para Cima/efeitos dos fármacos
10.
Biochim Biophys Acta ; 1833(12): 3054-3063, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23994619

RESUMO

MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of the HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto the HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin.


Assuntos
Sistema Hematopoético/citologia , Histona Acetiltransferases/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Linhagem Celular , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/genética , Humanos , Poliadenilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Immune Netw ; 23(4): e30, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37670813

RESUMO

About 0.8 million people die because of hepatitis B virus (HBV) infection each year. In around 5% of infected adults, the immune system is ineffective in countering HBV infection, leading to chronic hepatitis B (CHB). CHB is associated with hepatocellular carcinoma, which can lead to patient death. Unfortunately, although current treatments against CHB allow control of HBV infection, they are unable to achieve complete eradication of the virus. Cytokines of the IFN family represent part of the innate immune system and are key players in virus elimination. IFN secretion induces the expression of interferon stimulated genes, producing proteins that have antiviral properties and that are essential to cell-autonomous immunity. IFN-α is commonly used as a therapeutic approach for CHB. In addition, IFN-γ has been identified as the main IFN family member responsible for HBV eradication during acute infection. In this review, we summarize the key evidence gained from cellular or animal models of HBV replication or infection concerning the potential anti-HBV roles of IFN-γ with a particular focus on some IFN-γ-inducible genes.

12.
Pathog Dis ; 812023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-37012222

RESUMO

Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified.


Assuntos
Lipopolissacarídeos , Shigella flexneri , Humanos , Citosol/metabolismo , Citosol/microbiologia , Shigella flexneri/genética , Shigella flexneri/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo
13.
J Biol Chem ; 286(30): 26406-17, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21653699

RESUMO

The inhibitor of apoptosis protein cIAP1 (cellular inhibitor of apoptosis protein-1) is a potent regulator of the tumor necrosis factor (TNF) receptor family and NF-κB signaling pathways in the cytoplasm. However, in some primary cells and tumor cell lines, cIAP1 is expressed in the nucleus, and its nuclear function remains poorly understood. Here, we show that the N-terminal part of cIAP1 directly interacts with the DNA binding domain of the E2F1 transcription factor. cIAP1 dramatically increases the transcriptional activity of E2F1 on synthetic and CCNE promoters. This function is not conserved for cIAP2 and XIAP, which are cytoplasmic proteins. Chromatin immunoprecipitation experiments demonstrate that cIAP1 is recruited on E2F binding sites of the CCNE and CCNA promoters in a cell cycle- and differentiation-dependent manner. cIAP1 silencing inhibits E2F1 DNA binding and E2F1-mediated transcriptional activation of the CCNE gene. In cells that express a nuclear cIAP1 such as HeLa, THP1 cells and primary human mammary epithelial cells, down-regulation of cIAP1 inhibits cyclin E and A expression and cell proliferation. We conclude that one of the functions of cIAP1 when localized in the nucleus is to regulate E2F1 transcriptional activity.


Assuntos
Núcleo Celular/metabolismo , Ciclina A/biossíntese , Ciclina E/biossíntese , Fator de Transcrição E2F1/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologia , Animais , Núcleo Celular/genética , Proliferação de Células , Ciclina A/genética , Ciclina E/genética , Fator de Transcrição E2F1/genética , Inativação Gênica , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Camundongos , Estrutura Terciária de Proteína
14.
Nat Commun ; 12(1): 5862, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615873

RESUMO

NLRP3 controls the secretion of inflammatory cytokines IL-1ß/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.


Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Caseína Quinase II , Caseína Quinase Ialfa , Caspase 1/metabolismo , Citocinas/metabolismo , Enzimas Desubiquitinantes , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Quinases Relacionadas a NIMA/metabolismo , Fosforilação , Piroptose , Ubiquitinação
17.
Nat Commun ; 9(1): 242, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29339744

RESUMO

Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.


Assuntos
Caspases Iniciadoras/metabolismo , Caspases/metabolismo , Francisella/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Acilação , Animais , Caspases/genética , Caspases Iniciadoras/genética , Células Cultivadas , Citosol/microbiologia , Francisella/fisiologia , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interferência de RNA , Especificidade da Espécie , Células U937
18.
J Clin Invest ; 121(6): 2361-70, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21537084

RESUMO

Transcription intermediary factor 1γ (TIF1γ) was suggested to play a role in erythropoiesis. However, how TIF1γ regulates the development of different blood cell lineages and whether TIF1γ is involved in human hematological malignancies remain to be determined. Here we have shown that TIF1γ was a tumor suppressor in mouse and human chronic myelomonocytic leukemia (CMML). Loss of Tif1g in mouse HSCs favored the expansion of the granulo-monocytic progenitor compartment. Furthermore, Tif1g deletion induced the age-dependent appearance of a cell-autonomous myeloproliferative disorder in mice that recapitulated essential characteristics of human CMML. TIF1γ was almost undetectable in leukemic cells of 35% of CMML patients. This downregulation was related to the hypermethylation of CpG sequences and specific histone modifications in the gene promoter. A demethylating agent restored the normal epigenetic status of the TIF1G promoter in human cells, which correlated with a reestablishment of TIF1γ expression. Together, these results demonstrate that TIF1G is an epigenetically regulated tumor suppressor gene in hematopoietic cells and suggest that changes in TIF1γ expression may be a biomarker of response to demethylating agents in CMML.


Assuntos
Genes Supressores de Tumor , Leucemia Mielomonocítica Crônica/genética , Fatores de Transcrição/fisiologia , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Sequência de Bases , Diferenciação Celular , Metilação de DNA , Decitabina , Feminino , Regulação Leucêmica da Expressão Gênica , Hematopoese/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Leucemia Mielomonocítica Crônica/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Regiões Promotoras Genéticas , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Organismos Livres de Patógenos Específicos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
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