RESUMO
Although neonates are commonly exposed to vaginal herpes simplex virus (HSV)-2, neonatal herpes is rare. Therefore, we analyzed paired infant and maternal HSV-2 isolates from two cases of mother-to-infant transmission to identify viral factors contributing to vertical transmission. Sixteen infant isolates with neonatal herpes and 27 genital isolates in their third trimester were included. The infant isolates were significantly more temperature-independent than the maternal isolates. Sequence comparison revealed viral UL13 protein kinase (UL13-PK) mutation in the infant isolates in both cases. In the expanded cohort, infant isolates (5/18) had significantly more UL13-PK mutations than genital isolates (1/29). Isolates within 8 days post-birth (3/4) had a significantly higher frequency of UL13-PK mutation than those after 9 days (2/14), suggesting a close association between UL13-PK mutations and vertical transmission. Elongation factor 1-delta was identified as a target of UL13-PK by proteomic analysis of UL13-PK-positive and -negative HepG2 cells. The mixed infant isolates with the intact and mutated UL13-PK conferred altered cell tropism, temperature independence adapting to fetal temperature, and better growth properties in Vero and hepatoblastoma HepG2 cells than in HSV-2 with intact and mutated UL13-PK alone, indicating that viral UL13-PK mutation is essential for vertical HSV-2 transmission.
Assuntos
Herpes Simples , Complicações Infecciosas na Gravidez , Gravidez , Feminino , Recém-Nascido , Humanos , Herpesvirus Humano 2/genética , Mães , Proteômica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Virais/genética , Mutação , Tropismo , Transmissão Vertical de Doenças InfecciosasRESUMO
Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain-derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl-) cells through genome editing and compared HCV production between Abl- cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl-NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.
Assuntos
Hepacivirus , Proteínas Oncogênicas v-abl , Técnicas de Silenciamento de Genes , Células HEK293 , Hepacivirus/fisiologia , Hepatite C , Humanos , Proteínas Oncogênicas v-abl/genética , Proteínas Oncogênicas v-abl/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/genética , Replicação Viral/genéticaRESUMO
Systemic inflammatory response syndrome and sepsis are considered to contribute to hypercytokinemia in both patients with severe infection and immunocompromised condition. Past research has demonstrated that antibiotics and antifungals not only have antimicrobial efficacy but also affect the immune system. We previously examined whether immune cells were modulated by antibiotics such as tetracyclines or macrolides. The modulation of lipopolysaccharide-stimulated cells by those agents was elucidated. However, few reports about the modulation of the immune system by antifungal agents were found. In this study, the production of pro-inflammatory cytokines and chemokines and signaling pathways involved were investigated in zymosan-activated THP-1 cells. The effects were examined using antifungal agents such as echinocandin including caspofungin (CAS) and micafungin. Pro-inflammatory cytokine and chemokine levels were determined using enzyme-linked immunosorbent assay. Protein phosphorylation was evaluated by western blot analysis. CAS significantly decreased zymosan-induced pro-inflammatory cytokine and chemokine release in THP-1 cells. CAS (30 µg/mL) also downregulated tumor necrosis factor alpha levels, as shown by enzyme-linked immunosorbent assay. In western blot analysis, inhibitor of nuclear factor-kappa-B alpha, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and nuclear factor of activated T-cells phosphorylation and activation of caspase-1 and spleen tyrosine kinase (Syk) were downregulated. The major underlying mechanism of pro-inflammatory cytokine and chemokine suppression by CAS is to inhibit activation of Syk and its downstream signaling molecules. Based on the results, it can be concluded that CAS activity possibly involves Syk signaling pathways and has potential to prevent hypercytokinemia in fungal sepsis.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antifúngicos/farmacologia , Caspofungina/farmacologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Baço/enzimologia , Zimosan/farmacologia , Humanos , Transdução de Sinais , Células THP-1RESUMO
Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.
Assuntos
Cálcio/fisiologia , Receptor Muscarínico M3/fisiologia , Receptor PAR-2/fisiologia , Receptores Histamínicos H1/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células HT29 , Humanos , NF-kappa B/metabolismoRESUMO
There are many varieties of gastroenteritis viruses, of which norovirus (NoV) accounts for over 90% of the viral food poisoning incidents in Japan. However, protocols for rapidly identifying other gastroenteritis viruses need to be established to investigate NoV-negative cases intensively. In this study, a multiplex real-time PCR assay targeting rotavirus A, rotavirus C, sapovirus, astrovirus, adenovirus, and enterovirus was developed using stool samples collected from gastroenteritis patients between 2010 and 2013 in Fukui Prefecture, Japan. Of the 126 samples collected sporadically from pediatric patients with suspected infectious gastroenteritis, 51 were positive for non-NoV target viruses, whereas 27 were positive for NoV, showing a high prevalence of non-NoV viruses in pediatric patients. In contrast, testing in 382 samples of 58 gastroenteritis outbreaks showed that non-NoV viruses were detected in 13 samples, with NoV in 267. Of the 267 NoV-positive patients, only two were co-infected with non-NoV target viruses, suggesting that testing for non-NoV gastroenteritis viruses in NoV-positive samples was mostly unnecessary in outbreak investigations. Given these results, multiplex real-time PCR testing for non-NoV gastroenteritis viruses, conducted separately from NoV testing, may be helpful to deal with two types of epidemiological investigations, regular surveillance of infectious gastroenteritis and urgent testing when gastroenteritis outbreaks occur.
Assuntos
Gastroenterite/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vírus/isolamento & purificação , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Criança , Pré-Escolar , Coinfecção/epidemiologia , Coinfecção/virologia , Surtos de Doenças , Fezes/virologia , Doenças Transmitidas por Alimentos/diagnóstico , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/virologia , Gastroenterite/diagnóstico , Gastroenterite/epidemiologia , Humanos , Lactente , Japão/epidemiologia , Norovirus/genética , RNA Viral/genética , Rotavirus/genética , Rotavirus/isolamento & purificação , Sapovirus/genética , Sapovirus/isolamento & purificação , Vírus/classificação , Vírus/genéticaRESUMO
The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocinas/genética , Regulação da Expressão Gênica , Fagocitose , Receptores Fc/metabolismo , Quinase Syk/metabolismo , Tirosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Quimiocinas/metabolismo , Humanos , Mutação , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Fc/química , Receptores Fc/genética , Quinase Syk/química , Quinase Syk/genética , Células U937 , Domínios de Homologia de srcRESUMO
Infiltration by IgG-positive plasma cells is a common finding in tubulointerstitial nephritis. Indeed, it has been thought that CD138-positive mature plasma cells secrete mainly IgG, and the occurrence of tubulointerstitial nephritis with CD138-positive plasma cells secreting IgM has rarely been reported. Routine immunofluorescence of fresh frozen sections is considered the gold standard for detection of immune deposits. However, the immunoenzyme method with formalin-fixed, paraffin-embedded sections is superior for detecting IgM- or IgG-positive cells within the renal interstitium, thus histologic variants may often go undetected. We recently discovered a case of tubulointerstitial nephritis showing IgM-positive plasma cell accumulation within the interstitium. To further explore the morphologic and clinical features of such cases, we performed a nationwide search for patients with biopsy-proven tubulointerstitial nephritis and high serum IgM levels. We identified 13 patients with tubulointerstitial nephritis and IgM-positive plasma cell infiltration confirmed with the immunoenzyme method. The clinical findings for these patients included a high prevalence of distal renal tubular acidosis (100%), Fanconi syndrome (92%), and anti-mitochondrial antibodies (82%). The pathologic findings were interstitial nephritis with diffusely distributed CD3-positive T lymphocytes and colocalized IgM-positive plasma cells, as well as tubulitis with CD3-positive T lymphocytes in the proximal tubules and collecting ducts. Additionally, levels of H+-ATPase, H+, K+-ATPase, and the HCO3--Cl- anion exchanger were markedly decreased in the collecting ducts. We propose to designate this group of cases, which have a common histologic and clinical form, as IgM-positive plasma cell-tubulointerstitial nephritis.
Assuntos
Imunoglobulina M , Nefrite Intersticial/sangue , Nefrite Intersticial/imunologia , Plasmócitos/imunologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gαq/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gαq/11 dependent intracellular Ca2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gαq/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.
Assuntos
Receptores ErbB/genética , Receptor Muscarínico M3/genética , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Fosforilação , Receptor Muscarínico M3/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Interferon-α (IFN-α) and IFN-λ are structurally distinct cytokines that bind to different receptors, but induce expression of similar sets of genes through Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. The difference between IFN-α and IFN-λ signaling remains poorly understood. Here, using the CRISPR/Cas9 system, we examine the role of STAT1 and STAT2 in the inhibition of hepatitis C virus (HCV) replication by IFN-α and IFN-λ. Treatment with IFN-α increases expression of IFN-stimulated genes (ISGs) such as double-stranded RNA-activated protein kinase (PKR) and decreases viral RNA and protein levels in HCV-infected Huh-7.5 human hepatoma cells. These responses are only partially attenuated by knockout of STAT1 but are abolished by knockout of STAT2. In contrast, the inhibition of HCV replication by IFN-λ is abolished by knockout of STAT1 or STAT2. Microarray analysis reveals that IFN-α but not IFN-λ can induce expression of the majority of ISGs in STAT1 knockout cells. These findings suggest that IFN-α can inhibit HCV replication through a STAT2-dependent but STAT1-independent pathway, whereas IFN-λ induces ISG expression and inhibits HCV replication exclusively through a STAT1- and STAT2-dependent pathway.
Assuntos
Hepacivirus/genética , Interferon-alfa/genética , Interferon gama/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT2/genética , Antivirais/administração & dosagem , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Replicação do DNA/genética , Hepacivirus/patogenicidade , Hepatite C/genética , Hepatite C/patologia , Hepatite C/virologia , Humanos , Fator Regulador 1 de Interferon/genética , RNA Viral/genética , Replicação Viral/genéticaRESUMO
Impaired intestinal barrier function is one of the critical issues in inflammatory bowel diseases. The aim of this study is to investigate muscarinic cholinoceptor (mAChR)-mediated signaling for the amelioration of cytokine-induced barrier dysfunction in intestinal epithelium. Rat colon challenged with TNF-α and interferon γ reduced transepithelial electrical resistance (TER). This barrier injury was attenuated by muscarinic stimulation. In HT-29/B6 intestinal epithelial cells, muscarinic stimulation suppressed TNF-α-induced activation of NF-κB signaling and barrier disruption. Finally, muscarinic stimulation promoted the shedding of TNFR1, which would be a mechanism for the attenuation of TNF-α/NF-κB signaling and barrier disruption via mAChR.
Assuntos
Mucosa Intestinal/citologia , Receptores Muscarínicos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Colo/citologia , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , NF-kappa B/metabolismo , Ratos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330).
Assuntos
Hepacivirus/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Hepacivirus/genética , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Microscopia Confocal , Fosforilação , Proteínas Proto-Oncogênicas c-abl/genética , Interferência de RNA , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/genética , Tirosina/metabolismo , Proteínas não Estruturais Virais/genética , Vírion/genética , Vírion/metabolismo , Vírion/fisiologiaRESUMO
Dectin-1 recognizes ß-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity.
Assuntos
Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Mastócitos/enzimologia , Proteínas Tirosina Quinases/metabolismo , Animais , Antifúngicos/química , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Imunidade Inata , Macrófagos/metabolismo , Macrófagos/microbiologia , Mastócitos/citologia , Camundongos , Micoses/imunologia , Fosforilação , Ratos , Transdução de Sinais , Quinase Syk , Tirosina/química , beta-Glucanas/química , beta-Glucanas/metabolismoRESUMO
Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr(174), Tyr(183) and Tyr(446) in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr(183) and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr(174), Tyr(183) and Tyr(426) of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr(426) is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr(426) was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr(426) following BCR stimulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Células COS , Linhagem Celular , Galinhas , Chlorocebus aethiops , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação/fisiologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Quinase Syk , Domínios de Homologia de srcRESUMO
Non-receptor type of protein-tyrosine kinase Syk (spleen tyrosine kinase) was isolated in University of Fukui in 1991. Syk is most highly expressed by haemopoietic cells and known to play crucial roles in the signal transduction through various immunoreceptors of the adaptive immune response. However, recent reports demonstrate that Syk also mediates other biological functions, such as innate immune response, osteoclast maturation, platelet activation and cellular adhesion. Moreover, ectopic expression of Syk by epigenetic changes is reported to cause retinoblastoma. Because of its critical roles on the cellular functions, the development of Syk inhibitors for clinical use has been desired. Although many candidate compounds were produced, none of them had progressed to clinical trials. However, novel Syk inhibitors were finally developed and its usefulness has been evaluated in the treatment of allergic rhinitis, rheumatoid arthritis and idiopathic thrombocytopenic purpura. In this review, we will summarize the history, structure and function of Syk, and then the novel Syk inhibitors and their current status. In addition, we will introduce our research focused on the functions of Syk on Dectin-1-mediated mast cell activation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Aminopiridinas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mastócitos/enzimologia , Morfolinas , Oxazinas/uso terapêutico , Proteínas Tirosina Quinases/fisiologia , Piridinas/uso terapêutico , Pirimidinas , Quinase SykRESUMO
Non-receptor type of protein-tyrosine kinase Syk (spleen tyrosine kinase) was isolated in the University of Fukui in 1991. Syk is known to be essential for the various physiological functions, especially in hematopoietic lineage cells. Moreover, ectopic expression of Syk by epigenetic changes is reported to cause retinoblastoma. Recently, novel Syk inhibitors were developed and its usefulness has been evaluated in the treatment of allergic rhinitis, rheumatoid arthritis, and idiopathic thrombocytopenic purpura. In this review, we will summarize the history, structure, and function of Syk, and then describe the novel Syk inhibitors and their current status. Furthermore, we will introduce our findings of the adaptor protein 3BP2 (c-Abl SH3 domain-binding protein-2), as a novel target of Syk.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Tirosina Quinases/química , Transdução de Sinais/efeitos dos fármacos , Quinase SykRESUMO
Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.
Assuntos
Linfócitos B/enzimologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Ligantes , Fosforilação , Reação em Cadeia da Polimerase , Domínios de Homologia de srcRESUMO
Adaptor protein 3BP2, a c-Abl Src homology 3 (SH3) domain-binding protein, is tyrosine phosphorylated and positively regulates mast cell signal transduction after the aggregation of the high affinity IgE receptor (FcεRI). Overexpression of the Src homology 2 (SH2) domain of 3BP2 results in the dramatic suppression of antigen-induced degranulation in rat basophilic leukemia RBL-2H3 cells. Previously, a linker for activation of T cells (LAT) was identified as one of the 3BP2 SH2 domain-binding protein. In this report, to further understand the functions of 3BP2 in FcεRI-mediated activation of mast cell, we explored the protein that associates with the SH2 domain of 3BP2 and found that SH2 domain-containing phosphatase-1 (SHP-1) inducibly interacts with the SH2 domain of 3BP2 after the aggregation of FcεRI. The phosphorylation of Tyr(564) in the carboxy (C)-terminal tail region of SHP-1 is required for the direct interaction of SHP-1 to the SH2 domain of 3BP2. The expression of the mutant form of SHP-1 which was unable to interact with 3BP2 resulted in the significant reduction in SHP-1-mediated tumor necrosis factor-α (TNF-α) production without any effects on the degranulation in antigen-stimulated RBL-2H3 cells. These findings suggest that 3BP2 directly interacts with Tyr(564) -phosphorylated form of SHP-1 and positively regulates the function of SHP-1 in FcεRI-mediated signaling in mast cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hipersensibilidade/metabolismo , Mastócitos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Domínios de Homologia de src/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Alérgenos/administração & dosagem , Alérgenos/efeitos adversos , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Linhagem Celular Tumoral , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Espectrometria de Massas , Mastócitos/citologia , Mastócitos/imunologia , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/química , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Ratos , Receptores de IgE/imunologia , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/imunologia , Tirosina/metabolismo , Domínios de Homologia de src/imunologiaRESUMO
Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation.
Assuntos
Hepacivirus/fisiologia , Proteínas Quinases/metabolismo , Replicação Viral , Quinases Proteína-Quinases Ativadas por AMP , Linhagem Celular , Meios de Cultura/química , Glucose/metabolismo , Hepacivirus/crescimento & desenvolvimento , Hepatócitos/virologia , Humanos , Metformina/metabolismoRESUMO
Tyrosine phosphorylation of adaptor protein c-Abl-Src homology 3 (SH3) domain-binding protein-2 (3BP2, also referred to SH3BP2) positively regulates the B-cell antigen receptor (BCR)-mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B-cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2-P416R, corresponding to P418R in human protein) enhanced BCR-mediated activation of NFAT. 3BP2-P416R increased the signaling complex formation with Syk, phospholipase C-γ2 (PLC-γ2), and Vav1. In contrast, 3BP2-P416R could not change the association with the negative regulator 14-3-3. Loss of the association mutant that was incapable to associate with 14-3-3 could not mimic BCR-mediated NFAT activation in Syk-deficient cells. Moreover, BCR-mediated phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Querubismo/genética , Querubismo/metabolismo , Mutação Puntual/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas 14-3-3/metabolismo , Animais , Linfócitos B/metabolismo , Células COS , Linhagem Celular , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase 4/metabolismo , Complexos Multiproteicos/metabolismo , Fatores de Transcrição NFATC/genética , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Ativação Transcricional , Tirosina/metabolismoRESUMO
The palm fruit açaí is known to have potential health benefits due to its antioxidant scavenging capacities. Pretreatment of IgE-sensitized mouse primary cultured mast cells with açaí pulp resulted in the dramatic suppression of antigen-induced degranulation in a dose-dependent manner. Similarly, açaí suppressed IgE-mediated degranulation and transcription of the cytokine genes from a cultured mast cell line of rat basophilic leukemia (RBL)-2H3 cells. Açaí could selectively inhibit FcεRI signaling pathways. Furthermore, the FcεRI-mediated complementary signaling pathway was also suppressed by açaí. These results demonstrate that açaí is a potent inhibitor of IgE-mediated mast cell activation.