RESUMO
The inhibitory signaling of natural killer (NK) cells is crucial in the regulation of innate immune responses. Here we show that the association of KIR2DL1, an inhibitory receptor of NK cells, with beta-arrestin 2 mediated recruitment of the tyrosine phosphatases SHP-1 and SHP-2 to KIR2DL1 and facilitated 'downstream' inhibitory signaling. Consequently, the cytotoxicity of NK cells was higher in beta-arrestin 2-deficient mice but was inhibited in beta-arrestin 2-transgenic mice. Moreover, beta-arrestin 2-deficient mice were less susceptible than wild-type mice to mouse cytomegalovirus infection, an effect that was abolished by depletion of NK cells. Our findings identify a previously unknown mechanism by which the inhibitory signaling in NK cells is regulated.
Assuntos
Arrestinas/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais/imunologia , Animais , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , beta-Arrestina 2 , beta-ArrestinasRESUMO
MAP (Mitogen-activated protein) kinases play an important role in regulating many critical cellular processes. The inactivation of MAP kinases is always accomplished by a family of dual-specificity phosphatases, termed MAPK phosphatases (MKPs). Here, we have identified a novel MKP-like protein, designated DMKP-4, from the Drosophila genome. DMKP-4 is a protein of 387 amino acids, with a dual-specificity phosphatase (DSP) catalytic domain. Recombinant protein DMKP-4 retains intrinsic phosphatase activity against chromogenic substrate pNPP. Overexpression of DMKP-4 inhibited the activation of ERK, JNK and p38 by H(2)O(2), sorbitol and heat shock in HEK293-T cells, and JNK activation in Drosophila S2 cells under PGN stimuli. "Knockdown" of DMKP-4 expression by RNAi significantly enhanced the PGN-stimulated activation of JNK, but not ERK nor p38. Further study revealed that DMKP-4 interacted specifically with JNK via its DSP domain. Mutation of Cys-126 to serine in the DSP domain of DMKP-4 not only eliminated its interaction with JNK, but also markedly reduced its phosphatase activity. Thus, DMKP-4 is a Drosophila homologue of mammalian MKPs, and may play important roles in the regulation of various developmental processes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Fosfatases de Especificidade Dupla/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Compostos de Anilina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Cisteína/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/genética , Ativação Enzimática , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Compostos Organofosforados/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Mitogen-activated protein (MAP) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. MAP phosphatases (MKP)-1 is an archetypical member of the dual-specificity phosphatase family that deactivates MAP kinases. Induction of MKP-1 has been implicated in attenuating the lipopolysaccharide (LPS) and Peptidoglycan (PGN) responses, but how the expression of the MKP-1 is regulated is still not fully understood. Here, we show that inhibition of p38 MAP kinase by specific inhibitor SB 203580 or RNA interference (RNAi) markedly reduced the expression of MKP-1 in LPS or PGN-treated macrophages, which is correlated with prolonged activation of p38 and JNK. Depletion of MAPKAP kinase 2 (MK2), a downstream substrate of p38, by RNAi also inhibited the expression of MKP-1. The mRNA level of MKP-1 is not affected by inhibition of p38, but the expression of MKP-1 is inhibited by treatment of cycloheximide. Thus, p38 MAPK plays a critical role in mediating expression of MKP-1 at a post-transcriptional level. Furthermore, inhibition of p38 by SB 203580 prevented the expression of MKP-1 in LPS-tolerized macrophages, restored the activation of MAP kinases after LPS restimulation. These results indicate a critical role of p38-MK2-dependent induction of MKP-1 in innate immune responses.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Retroalimentação Fisiológica , Regulação Enzimológica da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Macrófagos/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Interações Medicamentosas , Tolerância a Medicamentos , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Peptidoglicano/farmacologia , Biossíntese de Proteínas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases , Piridinas/farmacologia , Interferência de RNA , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
This research aimed to evaluate the potential of Cordyceps sobolifera in mycelial biomass production via liquid culture and to assay the safety and determine the antioxidative and antiaging activities of Caenorhabditis elegans. A C. sobolifera isolate was cultured using the one-factor-at-a-time method to illustrate its carbon and nitrogen requirements. To assess safety, we determined the lethality, locomotion behavior, and reproduction of C. elegans cultured on a mycelial water extract (MWE) containing nematode growth medium (NGM). To investigate antiaging activity, C. elegans treated with MWE was incubated on NGM plates. The lethality was recorded throughout the whole life cycle. To identify antioxidant activity, C. elegans treated with MWE was exposed to paraquat, causing superoxide conditions. The results showed that C. sobolifera was favored by glucose and peptone as carbon and nitrogen sources, respectively. MWE was considered to be safe, as no abnormal behaviors were observed in C. elegans. Compared with nematodes pretreated with no MWE but with water instead, MWE at 1.0 mg/mL significantly prolonged the mean lifespan of C. elegans by 24%. We observed an obvious dose-effect relation between concentration and mean lifespan. The effective antioxidant activity was recorded at the high concentration of MWE. These findings demonstrate the potential antiaging and antioxidant properties of C. sobolifera as functional food and dietary supplement.
Assuntos
Antioxidantes/farmacologia , Caenorhabditis elegans/microbiologia , Cordyceps/química , Micélio/química , Animais , Biomassa , Caenorhabditis elegans/fisiologia , Cordyceps/fisiologia , Meios de Cultura , Técnicas de Cultura , Fermentação , Glucose/metabolismo , Estágios do Ciclo de Vida/efeitos dos fármacos , Micélio/fisiologia , Peptonas/metabolismo , Fatores de Tempo , Água/químicaRESUMO
The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.
Assuntos
Drosophila/metabolismo , MAP Quinase Quinase 2/metabolismo , MAP Quinase Quinase 3/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Drosophila/efeitos dos fármacos , Drosophila/efeitos da radiação , Temperatura Alta , MAP Quinase Quinase 1/efeitos dos fármacos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 1/efeitos da radiação , MAP Quinase Quinase 2/efeitos dos fármacos , MAP Quinase Quinase 2/efeitos da radiação , MAP Quinase Quinase 3/efeitos dos fármacos , MAP Quinase Quinase 3/efeitos da radiação , MAP Quinase Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Peptidoglicano/farmacologia , Interferência de RNA , RNA de Cadeia Dupla/farmacologia , Transdução de Sinais , Cloreto de Sódio/farmacologia , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos da radiaçãoRESUMO
The TAK1 plays a pivotal role in the innate immune response of Drosophila by controlling the activation of JNK and NF-kappaB. Activation of TAK1 in mammals is mediated by two TAK1-binding proteins, TAB1 and TAB2, but the role of the TAB proteins in the immune response of Drosophila has not yet been established. Here, we report the identification of a TAB2-like protein in Drosophila called dTAB2. dTAB2 can interact with dTAK1, and stimulate the activation of the JNK and NF-kB signaling pathway. Furthermore, we have found that silencing of dTAB2 expression by dsRNAi inhibits JNK activation by peptidoglycans (PGN), but not by NaCl or sorbitol. In addition, suppression of dTAB2 blocked PGN-induced expression of antibacterial peptide genes, a function normally mediated by the activation of NF-kappaB signaling pathway. No significant effect on p38 activation by dTAB2 was found. These results suggest that dTAB2 is specifically required for PGN-induced activation of JNK and NF-kappaB signaling pathways.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Animais , Células Cultivadas , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Ativação Enzimática , Imunidade Inata , Proteínas de Insetos/fisiologia , NF-kappa B/farmacologia , Peptidoglicano/farmacologia , Ligação Proteica , Interferência de RNA , Transdução de Sinais , Cloreto de Sódio/farmacologia , Sorbitol/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Impatiens balsamina L. (Balsaminaceae), an annual herb found throughout China, has been extensively used in traditional Chinese medicine (TCM). However, our knowledge regarding the adverse effects of I. balsamina in vivo is very limited. In this present study, the nematode Caenorhabditis elegans model was employed to fully assess the adverse effects of hydroalcoholic (EtOH 55%) extracts of I. balsamina stems (HAEIBS) in vivo. After exposure to 10 mg/mL HAEIBS, the major organism-level endpoints of C. elegans of percent survival, frequency of head thrash and body bends, and reproduction had decreased by 24%, 30%, and 25%, respectively. The lifespan of C. elegans was also greatly reduced after HAEIBS exposure compared to the controls. The active compounds in HAEIBS were separated using high speed countercurrent chromatograph (HSCCC) and characterized by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Two compounds, lawsone and 2-methoxy-1,4-naphthoquinone (MNQ), and their adverse effects were then more thoroughly detailed in this study. It was found that lawsone is the major toxin in HAEIBS with a higher toxicity than MNQ in terms of negative impact on C. elegans mortality, locomotion, reproduction, and lifespan. Our data also suggests that the C. elegans model may be useful for assessing the possible toxicity of other Chinese medicines, plant extracts, and/or compounds.