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1.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723037

RESUMO

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).


Assuntos
Acil-Butirolactonas/metabolismo , Apoptose , Bactérias/imunologia , Bactérias/metabolismo , Imunomodulação , Transdução de Sinais , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Fenômenos Fisiológicos Bacterianos , Cromatografia Líquida , Humanos , Vigilância Imunológica , Espectrometria de Massas , Proteômica/métodos
2.
Nat Immunol ; 17(3): 250-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26642356

RESUMO

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1ß (IL-1ß) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.


Assuntos
Proteínas de Transporte/imunologia , Macrófagos/imunologia , Mitose/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Caspase 1 , Cromatografia em Gel , Ensaio de Unidades Formadoras de Colônias , Citocinas , Proteínas do Citoesqueleto , Células Dendríticas , Encefalomielite Autoimune Experimental/imunologia , Feminino , Citometria de Fluxo , Células HEK293 , Humanos , Imunoprecipitação , Técnicas In Vitro , Inflamassomos/genética , Inflamassomos/imunologia , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Monócitos , Quinases Relacionadas a NIMA , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio , Medula Espinal/imunologia
3.
ACS Chem Biol ; 8(6): 1117-20, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23517377

RESUMO

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells over normal cells; however, tumor cells may develop TRAIL resistance. Here, we demonstrate that this resistance can be overcome in the presence of bacterial acylhomoserine lactones (AHLs) or AHL-producing bacteria through the combined effect of TRAIL-induced apoptosis and AHL-mediated inhibition of inflammation regulated by NF-κB signaling. This discovery unveils a previously unrecognized symbiotic link between bacteria and host immunosurveillance.


Assuntos
Acil-Butirolactonas/imunologia , Citocinas/imunologia , Neoplasias/imunologia , Neoplasias/microbiologia , Pseudomonas aeruginosa/imunologia , Acil-Butirolactonas/química , Apoptose , Linhagem Celular Tumoral , Humanos , NF-kappa B/imunologia , Pseudomonas aeruginosa/química , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/imunologia
4.
Science ; 340(6133): 711-6, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23539181

RESUMO

Vaccine development to induce broadly neutralizing antibodies (bNAbs) against HIV-1 is a global health priority. Potent VRC01-class bNAbs against the CD4 binding site of HIV gp120 have been isolated from HIV-1-infected individuals; however, such bNAbs have not been induced by vaccination. Wild-type gp120 proteins lack detectable affinity for predicted germline precursors of VRC01-class bNAbs, making them poor immunogens to prime a VRC01-class response. We employed computation-guided, in vitro screening to engineer a germline-targeting gp120 outer domain immunogen that binds to multiple VRC01-class bNAbs and germline precursors, and elucidated germline binding crystallographically. When multimerized on nanoparticles, this immunogen (eOD-GT6) activates germline and mature VRC01-class B cells. Thus, eOD-GT6 nanoparticles have promise as a vaccine prime. In principle, germline-targeting strategies could be applied to other epitopes and pathogens.


Assuntos
Vacinas contra a AIDS/imunologia , Células Germinativas/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Vacinas contra a AIDS/química , Vacinas contra a AIDS/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Antígenos CD4/imunologia , Cristalografia por Raios X , Análise Mutacional de DNA , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Humanos , Macaca , Camundongos , Modelos Animais , Dados de Sequência Molecular , Nanopartículas , Engenharia de Proteínas , Estrutura Terciária de Proteína
5.
Bioorg Med Chem Lett ; 22(5): 2043-5, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22300658

RESUMO

Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein complexes at the cytoplasmic domains of membrane-associated receptors in a stimulus-specific manner. Here, using the paradigm of cellular activation through cytokine and innate immune receptors, we demonstrate the proof-of-principle application of small molecule probes for the dissection of receptor-proximal signaling processes, such as activation of the transcription factor NF-κB and the protein kinase p38.


Assuntos
NF-kappa B/imunologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Células Cultivadas , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Bibliotecas de Moléculas Pequenas/química
6.
J Immunol ; 185(10): 6277-85, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20962258

RESUMO

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1ß and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3(-/-) and Asc(-/-) mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1ß, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.


Assuntos
Injúria Renal Aguda/metabolismo , Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Inflamassomos/metabolismo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/imunologia , Animais , Apoptose/imunologia , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/imunologia , Caspase 1/imunologia , Caspase 1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Inflamassomos/imunologia , Túbulos Renais , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Traumatismo por Reperfusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
J Immunol ; 184(5): 2297-304, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20124104

RESUMO

Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are members of a family of intracellular innate sensors that participate in innate immune responses to pathogens and molecules released during the course of tissue injury, including injury induced by ischemia. Ischemic injury to the kidney is characterized by renal tubular epithelial apoptosis and inflammation. Among the best studied intracellular innate immune receptors known to contribute to apoptosis and inflammation are Nod1 and Nod2. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Nod1, Nod2, Nod(1 x 2), and in their downstream signaling molecule receptor-interacting protein 2. We found that Nod1 and Nod2 were present in renal tubular epithelial cells in both mouse and human kidneys and that the absence of these receptors in mice resulted in protection from kidney ischemia reperfusion injury. Significant protection from kidney injury was seen with a deficiency of Nod2 and receptor-interacting protein 2, and the simultaneous deficiency of Nod1 and Nod2 provided even greater protection. We conclude that the intracellular sensors Nod1 and Nod2 play an important role in the pathogenesis of acute ischemic injury of the kidney, although possibly through different mechanisms.


Assuntos
Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Túbulos Renais/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Animais , Apoptose/genética , Apoptose/fisiologia , Transplante de Medula Óssea , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Rim/irrigação sanguínea , Rim/metabolismo , Túbulos Renais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Quimeras de Transplante/sangue , Quimeras de Transplante/genética
8.
Science ; 321(5886): 259-63, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18566250

RESUMO

The control of innate immune responses through activation of the nuclear transcription factor NF-kappaB is essential for the elimination of invading microbial pathogens. We showed that the bacterial N-(3-oxo-dodecanoyl) homoserine lactone (C12) selectively impairs the regulation of NF-kappaB functions in activated mammalian cells. The consequence is specific repression of stimulus-mediated induction of NF-kappaB-responsive genes encoding inflammatory cytokines and other immune regulators. These findings uncover a strategy by which C12-producing opportunistic pathogens, such as Pseudomonas aeruginosa, attenuate the innate immune system to establish and maintain local persistent infection in humans, for example, in cystic fibrosis patients.


Assuntos
4-Butirolactona/análogos & derivados , Regulação da Expressão Gênica , Homosserina/análogos & derivados , Macrófagos/imunologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais , 4-Butirolactona/fisiologia , Adulto , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fibrose Cística/microbiologia , Feminino , Homosserina/fisiologia , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Imunidade Inata , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa , Fosforilação , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/fisiologia , Receptores Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo
9.
Eur J Immunol ; 38(3): 788-96, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18266302

RESUMO

IRAK-4 kinase inactive (IRAK-4 KD) knock-in mice display defects in TLR- and IL-1 receptor signaling and are resistant to LPS-induced shock. In the present study we examined the LPS-induced response in IRAK-4 KD mice in more detail. We show that IRAK-4 kinase activity is required for certain aspects of TLR-mediated signaling but not for others. We found that IRAK-4 KD cells displayed reduced JNK and p38 signaling, while NF-kappaB was activated to a normal level but with delayed kinetics compared to wild-type cells. TLR4-mediated IRF3 activation was intact in these cells. Comprehensive analysis of expression of LPS-inducible genes by microarray demonstrated that IRAK-4 KD cells were severely impaired in the expression of many pro-inflammatory genes, suggesting their dependence on IRAK-4 kinase activity. In contrast, the expression of a subset of LPS-induced genes of anti-viral response was not affected by IRAK-4 kinase deficiency. Additionally, we demonstrate that LPS-activated early expression and production of some cytokines, e.g., TNF-alpha, is partially induced in the absence of IRAK-4 kinase activity. This suggests that the partially unaffected TLR4-mediated signaling could still drive expression of these genes in early phases and that IRAK-4 kinase activity is important for a more sustained anti-bacterial response.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Quinases Associadas a Receptores de Interleucina-1/genética , Lipopolissacarídeos/farmacologia , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Citocinas/sangue , Citocinas/genética , Citocinas/metabolismo , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Inibidor de NF-kappaB alfa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
10.
J Immunol ; 179(9): 6107-14, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17947685

RESUMO

Overactivation of the immune system upon acute bacterial infection leads to septic shock. Specific bacterial products potently stimulate immune cells via toll-like receptors (TLRs). Gram-negative bacteria induce a predominantly TLR4-driven signal through LPS release. To neutralize LPS signaling in experimental models of sepsis, we generated mAbs toward the TLR4/myeloid differentiation protein-2 (MD-2) complex. The binding properties of an array of selected rat mAbs differed in respect to their specificity for TLR4/MD-2 complex. The specificity of one such mAb, 5E3, to murine TLR4 was confirmed by its recognition of an epitope within the second quarter of the ectodomain. 5E3 inhibited LPS-dependent cell activation in vitro and prevented proinflammatory cytokine production in vivo following LPS challenge in a dose-dependent manner. Furthermore, 5E3 protected mice from lethal shock-like syndrome when applied using both preventative and therapeutic protocols. Most notably, in the colon ascendens stent peritonitis model of polymicrobial abdominal sepsis, administration of a single dose of 5E3 (50 mug) protected mice against mortality. These results demonstrate that neutralizing TLR4/MD-2 is highly efficacious in protecting against bacterial infection-induced toxemia and offers TLR4/MD-2 mAb treatment as a potential therapy for numerous clinical indications.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Choque Séptico/imunologia , Choque Séptico/terapia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Cricetinae , Cricetulus , Modelos Animais de Doenças , Humanos , Imunoterapia , Ligantes , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito , Camundongos , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismo , Taxa de Sobrevida , Receptor 4 Toll-Like/metabolismo
11.
Chem Biol ; 14(10): 1119-27, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17961824

RESUMO

Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors.


Assuntos
Anticorpos Monoclonais/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/efeitos dos fármacos , Aminobutiratos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Camundongos , Percepção de Quorum/genética , Percepção de Quorum/fisiologia , Transdução de Sinais/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Fatores de Virulência/genética
12.
J Bacteriol ; 189(18): 6619-25, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17631635

RESUMO

Chlamydia species are bacterial pathogens that affect over 140 million individuals worldwide. Ocular infection by Chlamydia trachomatis is the leading cause of preventable blindness, and urogenital tract infection by Chlamydia causes sexually transmitted disease. As obligate intracellular organisms, Chlamydia species have evolved mechanisms to evade the host immune system, including the degradation of the transcription factors regulatory factor X5 and upstream stimulation factor 1, which are required for the expression of major histocompatibility complex molecules I and II by CPAF and cleavage of p65 of the NF-kappaB pathway by the encoded CT441 protein. Here, we report the characterization of CT441 as a tail-specific protease. CT441 contains a PDZ domain of protein-protein interactions and a Ser/Lys dyad catalytic unit. Mutation at either Ser455 or Lys481 in the active site ablated CT441 activity of p65 cleavage. In addition, we found that the production of CT441 Tsp, which was detected at the middle and late stages of an infection, correlated with p65 cleavage activity. In addition to high homology, human and mouse p65 proteins also contain an identical C-terminal tail of 22 amino acid (aa) residues. However, only human p65 was susceptible to cleavage. Using molecular biology approaches, we mapped the p65 cleavage site(s) to a region that differs from that of mouse p65 by 6 aa residues. Additionally, the substitution of T352 with a proline inhibited p65 cleavage. Together, the study demonstrates that CT441 is a tail-specific protease that is capable of interfering with the NF-kappaB pathway of host antimicrobial and inflammatory responses.


Assuntos
Domínio Catalítico/genética , Chlamydia trachomatis/enzimologia , Chlamydia trachomatis/patogenicidade , Endopeptidases , Fator de Transcrição RelA/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Chlamydia trachomatis/genética , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Coelhos
13.
J Leukoc Biol ; 82(1): 177-83, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17403772

RESUMO

Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1beta, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP-induced IL-1beta release requires Nod2 and CIAS1/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, MDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-1beta production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Proteínas de Transporte/fisiologia , Interleucina-1beta/biossíntese , Proteína Adaptadora de Sinalização NOD2/fisiologia , Adjuvantes Imunológicos/farmacologia , Animais , Inflamação , Interleucina-6/biossíntese , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia
14.
J Biol Chem ; 282(18): 13552-60, 2007 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-17337443

RESUMO

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock-in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase was rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrate lack of cellular responsiveness to stimulation with IL-1beta or a Toll-like receptor 7 (TLR7) agonist. IRAK-4 kinase deficiency prevents the recruitment of IRAK-1 to the IL-1 receptor complex and its subsequent phosphorylation and degradation. IRAK-4 KD cells are severely impaired in NFkappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. As a consequence, IL-1 receptor/TLR7-mediated production of cytokines and chemokines is largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1 receptor (IL-1R)/TLR7-mediated induction of inflammatory responses.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/fisiologia , Receptor 7 Toll-Like/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/deficiência , Macrófagos/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Complexos Multiproteicos/genética , Mutação , Fosforilação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Interleucina-1/agonistas , Receptores de Interleucina-1/genética , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/genética
15.
J Biol Chem ; 281(45): 34592-600, 2006 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-16968706

RESUMO

Toll-like receptors (TLRs) play a crucial role in innate immunity by recognizing microbial pathogens. Triad3A is an E3 ubiquitin-protein ligase that interacts with the Toll/interleukin-1 receptor domain of TLRs and promotes their proteolytic degradation. In the present study, we further investigated its activity on signaling molecules downstream of TLRs and tumor necrosis factor (TNF) receptor 1. Triad3A promoted down-regulation of two TIR domain-containing adapter proteins, TIRAP and TRIF, as well as a RIP1 but had no effect on other adapter molecules in either the TLRs or TNF-alpha signaling pathways. Multiple sequence alignment analysis suggested that RIP1 contains a TIR homologous domain, and mutation of amino acid residues in this domain identified three residues critical for its interaction with Triad3A. Moreover, Triad3A acted as a negative regulator in TNF-alpha signaling. Reduction of Triad3A expression by small interference RNAs rendered cells hyperresponsive to TNF-alpha stimulation. Conversely, overexpression of Triad3A in cells blocked TNF-alpha-induced cell activation. This negative regulation was effected independently of changes in the cellular protein level of RIP1. Further studies indicated that RIP1 formed a complex with Triad3A and heat shock protein 90 (Hsp90), which is a chaperone protein capable of maintaining the stability of its client proteins. Treatment of cells with geldanamycin to disrupt the Hsp90 complex led to proteasomal degradation of RIP1. Depletion of Triad3A by small interference RNA treatment inhibited geldanamycin-activated ubiquitination and proteolytic degradation of RIP1. These results suggest that Triad3A is an E3 ubiquitin-protein ligase to RIP1 and that Hsp90 and Triad3A cooperatively maintain the homeostasis of RIP1.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Benzoquinonas/farmacologia , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Choque Térmico HSP90/genética , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Rim/citologia , Rim/metabolismo , Lactamas Macrocíclicas/farmacologia , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Plasmídeos , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/genética , Receptores de Interleucina-1/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases
16.
J Biol Chem ; 281(39): 28822-30, 2006 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-16893899

RESUMO

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). Here we have shown that p38 and eIF2alpha phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.


Assuntos
4-Butirolactona/análogos & derivados , Células da Medula Óssea/microbiologia , Regulação da Expressão Gênica , Macrófagos/microbiologia , 4-Butirolactona/química , 4-Butirolactona/fisiologia , Motivos de Aminoácidos , Animais , Células da Medula Óssea/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Pseudomonas aeruginosa/metabolismo , RNA Viral/metabolismo , Transdução de Sinais
17.
Nature ; 440(7087): 1064-8, 2006 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-16625199

RESUMO

Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.


Assuntos
Caspases/deficiência , Caspases/metabolismo , Listeria monocytogenes/imunologia , Sepse/imunologia , Sepse/microbiologia , Animais , Caspase 1/metabolismo , Caspase 12 , Inibidores de Caspase , Caspases/genética , Catálise , Linhagem Celular , Suscetibilidade a Doenças/enzimologia , Suscetibilidade a Doenças/imunologia , Suscetibilidade a Doenças/microbiologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-1/biossíntese , Interleucina-1/imunologia , Interleucina-1/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/fisiologia , Camundongos , Camundongos Knockout , Mutação , Peritonite/enzimologia , Peritonite/imunologia , Peritonite/microbiologia , Ligação Proteica , Sepse/enzimologia , Choque Séptico/enzimologia , Choque Séptico/imunologia , Choque Séptico/microbiologia , Taxa de Sobrevida
18.
Infect Immun ; 74(4): 2121-7, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16552041

RESUMO

The innate immune system surveys the extra- and intracellular environment for the presence of microbes. Among the intracellular sensors is a protein known as Nod2, a cytosolic protein containing a leucine-rich repeat domain. Nod2 is believed to play a role in determining host responses to invasive bacteria. A key element in upregulating host defense involves activation of the NF-kappaB pathway. It has been suggested through indirect studies that NF-kappaB-inducing kinase, or NIK, may be involved in Nod2 signaling. Here we have used macrophages derived from primary explants of bone marrow from wild-type mice and mice that either bear a mutation in NIK, rendering it inactive, or are derived from NIK-/- mice, in which the NIK gene has been deleted. We show that NIK binds to Nod2 and mediates induction of specific changes induced by the specific Nod2 activator, muramyl dipeptide, and that the role of NIK occurs in settings where both the Nod2 and TLR4 pathways are activated by their respective agonists. Specifically, we have linked NIK to the induction of the B-cell chemoattractant known as BLC and suggest that this chemokine may play a role in processes initiated by Nod2 activation that lead to improved host defense.


Assuntos
Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular , Células Cultivadas , Quimiocina CXCL13 , Quimiocinas CXC/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteína Adaptadora de Sinalização NOD2 , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Quinase Induzida por NF-kappaB
19.
Proc Natl Acad Sci U S A ; 103(6): 1840-5, 2006 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-16446438

RESUMO

Nod1, a cytosolic protein that senses meso-diaminopimelic acid-containing ligands derived from peptidoglycan, plays a role in host responses to invasive bacteria. Here we describe a function for Nod1, whereby it controls tumor formation. Cell lines derived from the human breast cancer epithelial cell line MCF-7 were used in a severe combined immune deficiency (SCID) mouse xenograft model to characterize a pathway linking Nod1 to the growth of estrogen-sensitive tumors. In MCF-7 cells, the absence of Nod1 correlates with tumor growth, an increased sensitivity to estrogen-induced cell proliferation, and a failure to undergo Nod1-dependent apoptosis. Conversely, overexpression of Nod1 in MCF-7 cells results in inhibition of estrogen-dependent tumor growth and reduction of estrogen-induced proliferative responses in vitro.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Neoplasias/genética , Proteína Adaptadora de Sinalização NOD1 , Fator de Necrose Tumoral alfa/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
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