RESUMO
Intestinal macrophages with functional plasticity play essential roles in gut immune responses by increasing chemokines and cytokines, thereby contributing to the pathogenesis of inflammatory bowel disease (IBD). Poly(rC)-binding protein 1 (PCBP1), which is widely expressed in immune cells, binds to nucleic acids in mRNA processing, stabilization, translation and transcription. However, little is known about the influence of PCBP1 on macrophages and its specific mechanism in inflamed intestines. In this study, conditional depletion of Pcbp1 in macrophages protected mice from progression of dextran sulfate sodium induced colitis and resulted in significant alleviation of colitis. Pcbp1 deficiency markedly decreased C-C motif chemokine ligand 2 (CCL2) production by colonic CX3C motif chemokine receptor 1+ (CX3CR1+) macrophages and reduced accumulation of pro-inflammatory macrophages and production of pro-inflammatory cytokines, such as IL-6 and TNF-α, in the inflamed colon. RNA-immunoprecipitation analysis indicated that PCBP1 might interact with Ccl2 mRNA and regulate its expression in macrophages. PCBP1 expression in inflamed intestines also correlated significantly with IBD severity in patients, suggesting a critical involvement of PCBP1 in intestinal inflammation. We anticipate that our findings will facilitate the development of novel therapeutic approaches for IBD by targeting the specific function of immune cells in the local microenvironment, thereby helping to reduce adverse effects.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Ligantes , Macrófagos , Colo , Quimiocinas , Citocinas/metabolismo , RNA Mensageiro/metabolismo , Sulfato de Dextrana/farmacologia , Camundongos Endogâmicos C57BLRESUMO
Interferon α (IFNα) is a type I interferon, an essential cytokine employed by the immune system to fight viruses. Although a number of the structures of type I interferons have been reported, most of the known structures of IFNα are in complex with its receptors. There are only two examples of structures of free IFNα: one is a dimeric X-ray structure without side-chain information; and another is an NMR structure of human IFNα. Although we have shown that Sortilin is involved in the secretion of IFNα, the details of the molecular interaction and the secretion mechanism remain unclear. Recently, we solved the X-ray structure of mouse Sortilin, but the structure of mouse IFNα remained unknown. In this study, we determined the crystal structure of mouse IFNα2 at 2.1 Å resolution and investigated its interaction with Sortilin. Docking simulations suggested that Arg22 of mouse IFNα2 is important for the interaction with mouse Sortilin. Mutation of Arg22 to alanine facilitated IFNα2 secretion, as determined by flow cytometry, highlighting the contribution of this residue to the interaction with Sortilin. These results suggest an important role for Arg22 in mouse IFNα for Sortilin-mediated IFNα trafficking.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Interferon-alfa/química , Interferon-alfa/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Animais , Antivirais/química , Antivirais/metabolismo , Cromatografia em Gel/métodos , Cristalografia por Raios X/métodos , Humanos , Interferon-alfa/genética , Camundongos , Simulação de Acoplamento Molecular/métodos , Mutação , Ligação Proteica , Transporte ProteicoRESUMO
Post-transcriptional regulation of cytokine production is crucial to ensure appropriate immune responses. We previously demonstrated that poly-rC-binding protein-1 (PCBP1) can act as a trans-acting factor to stabilize transcripts encoding sortilin, which mediates cytokine trafficking. Here, we report that PCBP2, which strongly resembles PCBP1, can stabilize sortilin transcripts in macrophages using the same mechanism employed by PCBP1. PCBP2 recognized the C-rich element in the 3' UTR of sortilin mRNA, and PCBP2 knockdown decreased sortilin transcripts, indicating that PCBP2 stabilizes sortilin mRNA by binding to its 3' UTR. Zn2+ reversibly inhibited the nucleotide binding ability of PCBP2 in vitro. These findings suggest that both PCBP2 and PCBP1 may control the stability of sortilin transcripts by sensing intracellular Zn2+ levels in immune cells.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Técnicas de Cultura de Células , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Zinco/metabolismoRESUMO
Sortilin is a multifunctional sorting receptor involved in cytokine production in immune cells. To understand the mechanism of Sortilin-mediated cytokine trafficking, we determined the 2.45-Å structure of the dimerized Sortilin ectodomain (sSortilin or the Vps10-domain) crystallized at acidic pH. Substantial conformational changes upon dimerization lead to the intermolecular hydrophobic interaction between the conserved E455 and F137. Analysis of the electrostatic surface and size-exclusion chromatography revealed that sSortilin dimerization occurs due to an increase in hydrophobic interactions at the neutral dimer interface at acidic pH. The N682-attached N-glycan in the vicinity of the dimer interface implies its involvement in the dimerization. The disruption of Sortilin dimerization by mutations impairs efficient interferon-alpha secretion from cells. These results suggest the functional importance of Sortilin dimerization in cytokine trafficking.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ácidos/farmacologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Interferon-alfa/metabolismo , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Estrutura Quaternária de Proteína/genética , Transporte Proteico/genéticaRESUMO
BACKGROUND/AIM: There exist few research articles regarding the anticancer activity of azulene-related compounds. We investigated here the relative cytotoxicity of 10 azulene amide derivatives against cancer and normal cells. MATERIALS AND METHODS: Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and three human oral normal cells (gingival fibroblasts, periodontal ligament fibroblasts and pulp cells) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide method. Antitumor activity was evaluated by tumor-specificity (TS) (ratio of mean 50% cytotoxic concentration (CC50) against normal cells to that against OSCC cell lines) and potency-selectivity expression (PSE) (ratio of TS to CC50 against tumor cells). Apoptosis-inducing activity was evaluated by cleavage of poly ADP-ribose polymerase and caspase-3 with western blot analysis. RESULTS: N-Propylguaiazulenecarboxamide [1] showed the highest TS and PSE values, compared to that of doxorubicin, and induced apoptosis in two OSCC cell lines. QSAR analysis demonstrated that their tumor-specificity of azulene amide derivatives was correlated with hydrophobicity and molecular shape. CONCLUSION: Compound [1] can be considered as a lead compound for manufacturing new anticancer drug candidates.
Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Azulenos , Amidas/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Azulenos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Estrutura MolecularRESUMO
Mast cells, basophils, and eosinophils are central effectors in allergic inflammatory disorders. These cells secrete abundant serine proteases as well as chemical mediators and cytokines; however, the expression profiles and functions of their endogenous inhibitors remain elusive. We found that murine secretory leukoprotease inhibitor (SLPI) is expressed in basophils and eosinophils but in not in mast cells. SLPI-deficient (Slpi-/-) basophils produce more cytokines than wild-type mice after IgE stimulation. Although the deletion of SLPI in basophils did not affect the release of chemical mediators upon IgE stimulation, the enzymatic activity of the serine protease tryptase was increased in Slpi-/- basophils. Mice transferred with Slpi-/- basophils were highly sensitive to IgE-mediated chronic allergic inflammation. Eosinophils lacking SLPI showed greater interleukin-6 secretion and invasive activity upon lipopolysaccharide stimulation, and the expression of matrix metalloproteinase-9 by these eosinophils was increased without stimulation. The absence of SLPI increases JNK1 phosphorylation at the steady state, and augments the serine phosphorylation of JNK1-downstream ETS transcriptional factor Elk-1 in eosinophils upon stimulation. Of note, SLPI interacts with a scaffold protein, JNK-interacting protein 3 (JIP3), that constitutively binds to the cytoplasmic domain of toll-like receptor (TLR) 4, suggesting that SLPI controls Elk-1 activation via binding to JIP3 in eosinophils. Mice transferred with Slpi-/- eosinophils showed the exacerbation of chitin-induced allergic inflammation. These findings showed that SLPI is a negative regulator in allergic effector cells and suggested a novel inhibitory role of SLPI in the TLR4 signaling pathways.
RESUMO
Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of arginine in protein molecules to a citrulline residue in a Ca(2+)-dependent manner. In this study, we determined the structure of an active form of human PAD1 crystallized in the presence of Ca(2+) at 3.2-Å resolution. Although human PAD2 and PAD4 isozymes were previously reported to form a head-to-tail homodimer, it is still unknown whether this quaternary structure is common to other PAD isozymes. The asymmetric unit of the crystal contained two PAD1 molecules; however, the head-to-tail dimeric form was not found. Small-angle X-ray scattering analyses revealed PAD1 to be a monomer in solution, while PAD3 was dimerized with a structure similar to PAD2 and PAD4. PAD1 was apparently different from the crystal structures of PAD2 and PAD4, with an elongated N-terminal loop that appears to prevent the formation of the homodimer. Of interest, the N-terminal loop occupied the substrate binding site of the adjacent PAD1 molecules in the crystal. Deimination of S100A3 peptides in vitro implied that PAD isozymes recognize the quaternary structure of S100A3. The substrate-accessible monomeric structure brought about by the extension of its N terminus may partly account for the highest tolerant substrate recognition of PAD1. This is the first ever report on the molecular structure of PAD1 demonstrating the unique monomeric form of the PAD isozyme.
Assuntos
Hidrolases/química , Arginina/química , Sítios de Ligação/fisiologia , Cristalografia por Raios X/métodos , Humanos , Isoenzimas/química , Proteína-Arginina Desiminase do Tipo 1 , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Raios XRESUMO
Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin plays a key role in cytokine production. We observed interactions of sortilin with multiple cytokines including IFN-α, and sortilin depletion in plasmacytoid dendritic cells (pDCs) led to a reduction of IFN-α secretion, suggesting a pivotal role of sortilin in the exocytic trafficking of IFN-α in pDCs. Moreover, sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Poly-rC-binding protein 1 (PCBP1) recognized the C-rich element (CRE) in the 3' UTR of sortilin mRNA, and depletion of PCBP1 enhanced the degradation of sortilin transcripts, suggesting that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. The nucleotide-binding ability of PCBP1 was impaired by zinc ions and alterations of intracellular zinc affect sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Células Dendríticas/imunologia , Interferon-alfa/imunologia , Plasmócitos/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células Dendríticas/citologia , Feminino , Células HEK293 , Humanos , Interferon-alfa/genética , Camundongos , Plasmócitos/citologia , Células RAW 264.7 , Transdução de Sinais/genética , Receptores Toll-Like/genéticaRESUMO
OBJECTIVES AND DESIGN: Hypersensitivity pneumonitis (HP) is a pulmonary disease caused by repeated exposure to various aspiration antigens, including bacteria and fungi. Although TLRs are known to be required for the generation of HP triggered by bacteria, the significance of fungal receptors remains unclear. The present study aimed to investigate whether Dectin-1 and Dectin-2 contribute to the development of experimental HP triggered by the fungus Trichosporon asahii (T. asahii) that causes summer-type HP. MATERIALS AND METHODS: We investigated the binding between Dectin-Fc protein and T. asahii by a dot blot assay. We performed the histological and flow cytometric analysis in the HP model using Dectin-1-deficient (Dectin-1(-/-)) and Dectin-2(-/-) mice. We also investigated Th17/Th1 responses in lung cells, and measured an IL-17-promoting cytokine IL-23 from bone marrow-derived dendritic cells (BMDCs) by ELISA. RESULTS: Dectin-1 bound more strongly to T. asahii than Dectin-2. Dectin-1(-/-) mice barely developed HP, whereas both wild-type mice and Dectin-2(-/-) mice developed similar lung diseases. Dectin-1 deficiency decreased the infiltration of neutrophils and monocyte-derived macrophages and repressed the expansion of lung CD4(+)IL-17A(+) cells. The production of IL-23 p19 was reduced in Dectin-1(-/-) BMDCs. CONCLUSIONS: These data suggested Dectin-1 plays a critical role in the development of fungus-induced HP.
Assuntos
Alveolite Alérgica Extrínseca/imunologia , Lectinas Tipo C/imunologia , Trichosporon , Alveolite Alérgica Extrínseca/patologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Interleucina-23/imunologia , Lectinas Tipo C/genética , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th17/imunologiaRESUMO
Plasmacytoid dendritic cells (PDCs) produce type I interferons (IFNs) in response to viral nucleic acids to exert antiviral immunity. However, PDCs are related to the progress and severity of autoimmune diseases, such as systemic lupus erythematosus, because they respond to host DNA. Therefore, the regulation of PDC activation is critical for maintaining adequate immune responses. Here we show that an inhibitory major histocompatibility complex class I receptor, paired immunoglobulin-like receptor B (PIR-B), suppressed Fms-like tyrosine kinase 3 ligand-induced PDC differentiation in BM cells, as well as Toll-like receptor 9-mediated IFN-α production by PDCs, through the dephosphorylation of STAT1/STAT2. In particular, PIR-B inhibited IFN-α-mediated STAT phosphorylation, suggesting that PIR-B negatively regulates the positive feedback mechanism of IFN-α secretion triggered by Toll-like receptor 9. These results demonstrate a novel regulatory role for PIR-B in PDCs.