RESUMO
Deregulation of the TH17 subset of helper T cells is closely linked with immunological disorders and inflammatory diseases. However, the mechanism by which TH17 cells are regulated remains elusive. Here we found that the phosphatase DUSP2 (PAC1) negatively regulated the development of TH17 cells. DUSP2 was directly associated with the signal transducer and transcription activator STAT3 and attenuated its activity through dephosphorylation of STAT3 at Tyr705 and Ser727. DUSP2-deficient mice exhibited severe susceptibility to experimental colitis, with enhanced differentiation of TH17 cells and secretion of proinflammatory cytokines. In clinical patients with ulcerative colitis, DUSP2 was downregulated by DNA methylation and was not induced during T cell activation. Our data demonstrate that DUSP2 is a true STAT3 phosphatase that modulates the development of TH17 cells in the autoimmune response and inflammation.
Assuntos
Diferenciação Celular/imunologia , Fosfatase 2 de Especificidade Dupla/imunologia , Fator de Transcrição STAT3/imunologia , Células Th17/imunologia , Animais , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Metilação de DNA/imunologia , Sulfato de Dextrana , Fosfatase 2 de Especificidade Dupla/deficiência , Fosfatase 2 de Especificidade Dupla/genética , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Immunoblotting , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/imunologia , Ligação Proteica/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Células Th17/metabolismo , Tirosina/imunologia , Tirosina/metabolismoAssuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Substituição de Aminoácidos/imunologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Humanos , Cinética , Ligantes , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tirosina/imunologiaRESUMO
The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we found that signaling pathways dependent on the kinases Syk and Jnk were required for the activation of caspase-1 via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or Jnk abolished the formation of ASC specks without affecting the interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and Jnk-dependent manner, which suggested that Syk and Jnk are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 in mouse ASC was critical for speck formation and caspase-1 activation. Our results suggest that phosphorylation of ASC controls inflammasome activity through the formation of ASC specks.
Assuntos
Proteínas do Citoesqueleto/imunologia , Inflamassomos/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Proteínas Tirosina Quinases/imunologia , Animais , Proteínas Reguladoras de Apoptose , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Caspase 1/imunologia , Caspase 1/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Células HEK293 , Humanos , Immunoblotting , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nigericina/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fosforilação/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Quinase Syk , Tirosina/genética , Tirosina/imunologia , Tirosina/metabolismoRESUMO
The effectiveness of innate immune responses relies on an intricate balance between activation and regulation. TLR8, a member of the Toll-like receptor (TLR) family, plays a fundamental role in host defense by sensing viral single-stranded RNAs (ssRNAs). However, the molecular recognition and regulatory mechanism of TLR8 is not fully understood, especially in a whole-cell environment. Here, we engineer the first light-controllable TLR8 model by genetically encoding a photocaged tyrosine, NBY, into specific sites of TLR8. In the caged forms, the activity of TLR8 is masked but can be restored upon decaging by exposure to UV light. To explain the mechanism clearly, we divide the sites with light responsiveness into three groups. They can separately block the ligands that bind to the pockets of TLR8, change the interaction modes between two TLR8 protomers, and interfere with the interactions between TLR8 cytosolic domains with its downstream adaptor. Specifically, we use this chemical caging strategy to probe and evaluate the function of several tyrosine sites located at the interface of TLR8 homodimers with a previously unknown regulatory mode, which may provide a new strategy for TLR8 modulator development. Effects on downstream signaling pathways are monitored at the transcriptional and translational levels in various cell lines. By photoactivating specific cells within a larger population, this powerful tool can provide novel mechanistic insights, with potential in biotechnological and pharmaceutical applications.
Assuntos
Imunidade Inata/imunologia , Receptor 8 Toll-Like/imunologia , Tirosina/imunologia , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Processos Fotoquímicos , Tirosina/química , Tirosina/genéticaRESUMO
Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705-SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT-CTT intermolecular and the CTT-SH2 intramolecular interactions that support pY705-SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705-SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705-SH2 interaction. Thus, pS727 triggers pY705-SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation-inactivation for proper function of STAT3.
Assuntos
Fator de Transcrição STAT3/imunologia , Serina/imunologia , Tirosina/imunologia , Células Cultivadas , Células HEK293 , Células Hep G2 , Humanos , Fosforilação , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Domínios de Homologia de src/imunologiaRESUMO
Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.
Assuntos
Ceramidas/imunologia , Ceramidas/metabolismo , Hipersensibilidade/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Células Cultivadas , Dermatite/imunologia , Dermatite/metabolismo , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Mastócitos/patologia , Camundongos , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Tirosina/imunologia , Tirosina/metabolismoRESUMO
Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues, which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with the generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens.
Assuntos
Antígeno Carcinoembrionário/imunologia , Imunidade nas Mucosas/imunologia , Intestinos/imunologia , Linfócitos T/imunologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Animais , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Citoplasma/genética , Citoplasma/imunologia , Citoplasma/metabolismo , Homeostase , Imunidade nas Mucosas/genética , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Mucosa Intestinal/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Ativação Linfocitária , Metagenoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Isoformas de Proteínas , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T/metabolismo , Tirosina/genética , Tirosina/imunologia , Tirosina/metabolismoRESUMO
Despite remarkable progresses in vaccinology, therapeutic cancer vaccines have not achieved their full potential. We previously showed that an excessively long duration of Ag presentation critically reduced the quantity and quality of vaccination-induced T cell responses and subsequent antitumor efficacy. In this study, using a murine model and tumor cell lines, we studied l-tyrosine amino acid-based microparticles as a peptide vaccine adjuvant with a short-term Ag depot function for the induction of tumor-specific T cells. l-Tyrosine microparticles did not induce dendritic cell maturation, and their adjuvant activity was not mediated by inflammasome activation. Instead, prolonged Ag presentation in vivo translated into increased numbers and antitumor activity of vaccination-induced CD8+ T cells. Indeed, prolonging Ag presentation by repeated injection of peptide in saline resulted in an increase in T cell numbers similar to that observed after vaccination with peptide/l-tyrosine microparticles. Our results show that the duration of Ag presentation is critical for optimal induction of antitumor T cells, and can be manipulated through vaccine formulation.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Peptídeos/imunologia , Adjuvantes Imunológicos , Animais , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Inflamassomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Tirosina/imunologia , Vacinação/métodos , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
CD5 and CD6 are related surface receptors that limit and promote T-cell responses. Co-stimulatory effects of CD6 depend on binding a cell surface ligand, CD166, and recruitment of the intracellular adaptor proteins GADS and SLP-76 by C-terminal phosphotyrosines. We have continued to identify interactions of CD5 and CD6 to understand their roles in T-cell activation. In a screen to identify binding partners for peptides containing a cytoplasmic sequence, SDSDY conserved between CD5 and CD6, we identified ezrin radixin moesin (ERM) proteins, which link plasma membrane proteins to actin. Purified radixin FERM domain bound directly to CD5 and CD6 SDSDY peptides in a phosphorylation-dependent manner (KD = 0·5-2 µm) at 37°. In human T-cell blasts, mutation of the CD6 SDSDY sequence enhanced CD69 expression in response to CD3 monoclonal antibody. In this proximal readout, interactions of the SDSDY sequence were dominant compared with the C-terminal tyrosines of CD6. In contrast, in a more downstream readout, interleukin-2 expression, in response to immobilized CD3 and CD6 monoclonal antibodies, the C-terminal tyrosines were dominant. The data suggest that varying functional effects of CD6 and potentially CD5 depend on interactions of different cytoplasmic regions with the cytoskeleton and alter depending on the stimuli.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD5/imunologia , Proteínas de Ligação a DNA/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Actinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Membrana Celular/imunologia , Citoplasma/imunologia , Citoesqueleto/imunologia , Humanos , Fosforilação/imunologia , Ratos , Tirosina/imunologiaRESUMO
Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.
Assuntos
Acetaldeído/química , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Conformação Proteica , Animais , Sítios de Ligação/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Eritrócitos/imunologia , Feminino , Hemólise/imunologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Microscopia Eletrônica de Transmissão , Simulação de Acoplamento Molecular , Estresse Oxidativo/imunologia , Ligação Proteica/imunologia , Coelhos , Tirosina/imunologiaRESUMO
Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 ß, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.
Assuntos
Anticorpos Monoclonais/imunologia , Clorpirifos/análogos & derivados , Proteínas/análise , Tirosina/análogos & derivados , Tirosina/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Clorpirifos/química , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Camundongos , Estrutura Molecular , Peptídeos/análise , Peptídeos/química , Peptídeos/imunologia , Proteínas/química , Proteínas/imunologia , Proteólise , Espectrometria de Massas em Tandem , Tirosina/químicaRESUMO
The amino acid tyrosine forms cytotoxic amyloid-like fibrils by molecular self-assembly. However, the production of antibodies towards tyrosine assemblies, reflecting their presentation to the immune system, was not demonstrated yet. Here, we describe the production of antibodies that specifically recognize tyrosine in its fibrillated form. The antibodies were demonstrated to specifically bind self-assembled tyrosine, in contrast to its non-aggregated form or disintegrated fibrils. The antibodies could be used for immunostaining of tyrosine fibrils in cultured cells. Furthermore, confocal microscopy allowed a demonstration of the intracellular presence of the metabolite amyloids in a neuroblastoma cell model. Finally, pre-incubation of tyrosine fibrils with the antibodies resulted in significant reduction in their cytotoxicity. Taken together, we provide an experimental proof for the immunogenicity of tyrosine amyloid fibrillary assemblies. These specific antibodies against tyrosine structures could be further used as a research tool to study the dynamics, toxicity and cellular localization of the assemblies.
Assuntos
Amiloide/antagonistas & inibidores , Amiloide/imunologia , Anticorpos/imunologia , Anticorpos/farmacologia , Tirosina/imunologia , Amiloide/química , Formação de Anticorpos/imunologia , Humanos , Modelos Anatômicos , Conformação Molecular , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Transporte Proteico , Tirosina/químicaRESUMO
Protein tyrosine nitration by oxidative and nitrate stress is important in the pathogenesis of many inflammatory or aging-related diseases. Mass spectrometry analysis of protein nitrotyrosine is very challenging because the non-nitrated peptides suppress the signals of the low-abundance nitrotyrosine (NT) peptides. No validated methods for enrichment of NT-peptides are currently available. Here we report an immunoaffinity enrichment of NT-peptides for proteomics analysis. The effectiveness of this approach was evaluated using nitrated protein standards and whole-cell lysates in vitro. A total of 1881 NT sites were identified from a nitrated whole-cell extract, indicating that this immunoaffinity-MS method is a valid approach for the enrichment of NT-peptides, and provides a significant advance for characterizing the nitrotyrosine proteome. We noted that this method had higher affinity to peptides with N-terminal nitrotyrosine relative to peptides with other nitrotyrosine locations, which raises the need for future study to develop a pan-specific nitrotyrosine antibody for unbiased, proteome-wide analysis of tyrosine nitration. We applied this method to quantify the changes in protein tyrosine nitration in mouse lungs after intranasal poly(I:C) treatment and quantified 237 NT sites. This result indicates that the immunoaffinity-MS method can be used for quantitative analysis of protein nitrotyrosines in complex samples.
Assuntos
Peptídeos/química , Proteômica/métodos , Tirosina/análogos & derivados , Animais , Anticorpos/análise , Sítios de Ligação , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Poli I-C/farmacologia , Tirosina/análise , Tirosina/química , Tirosina/imunologia , Tirosina/metabolismoRESUMO
The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the initial, rate-limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1-dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine-based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP-1 and SHP-2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine-phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half-life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM-associated tyrosines with phospho-mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 - but not ITIM2 mutant - did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen-specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1-dependent events will occur in a local microenvironment.
Assuntos
Imunossupressores/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Tirosina/imunologia , Animais , Citocinas/imunologia , Células Dendríticas/imunologia , Feminino , Meia-Vida , Tolerância Imunológica/imunologia , Cinurenina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/imunologia , Domínios Proteicos/imunologia , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/imunologia , Triptofano/imunologiaRESUMO
The adaptor protein SLP76 directs signaling downstream of the T cell receptor (TCR) and is essential for thymocyte development. SLP76 contains three N-terminal tyrosines that are critical for its function. To define the role of these residues in thymocyte development, we generated two lines of "knock-in" mice, one expressing a mutation in tyrosine 145 (Y145F) and a second harboring two point mutations at tyrosines 112 and 128 (Y112-128F). We show here that although thymocyte development requires both Y145- and Y112-128-generated signals, selection was more dependent upon Y145. Although several proximal TCR signaling events were defective in both mutant mice, phosphorylation of the guanine nucleotide exchange factor, Vav1, and activation of Itk-dependent pathways were differentially affected by mutations at Y112-128 and Y145, respectively. Analysis of mice expressing one Y145F and one Y112-128F allele revealed that these mutants could complement one another in trans, demonstrating cooperativity between two or more SLP76 molecules. Thus, the N-terminal tyrosines of SLP76 are required for thymocyte selection but can function on separate molecules to support TCR signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Diferenciação Celular/imunologia , Fosfoproteínas/imunologia , Transdução de Sinais/imunologia , Linfócitos T/citologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Citometria de Fluxo , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Mutantes , Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Tirosina/química , Tirosina/genética , Tirosina/imunologiaRESUMO
IL-25 promotes type 2 immunity by inducing the expression of Th2-associated cytokines. Although it is known that the IL-25R (IL-17RB) recruits the adaptor protein ACT1, the IL-25R signaling mechanism remains poorly understood. While screening for IL-25R components, we found that IL-25 responses were impaired in Traf4 (-/-) cells. Administering IL-25 to Traf4 (-/-) mice resulted in blunted airway eosinophilia and Th2 cytokine production. Notably, IL-25R recruitment of TRAF4 was required for the ACT1/IL-25R interaction. Mechanistically, TRAF4 recruited the E3-ligase SMURF2, to degrade the IL-25R-inhibitory molecule DAZAP2. Silencing Dazap2 increased ACT1/IL-25R interaction and IL-25 responsiveness. Moreover, a tyrosine within the IL-25R elicited DAZAP2 interference. This study indicates that TRAF4-SMURF2-mediated DAZAP2 degradation is a crucial initiating event for the IL-25 response.
Assuntos
Proteínas de Transporte/imunologia , Inflamação/imunologia , Receptores de Interleucina/imunologia , Sistema Respiratório/imunologia , Fator 4 Associado a Receptor de TNF/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Immunoblotting , Inflamação/genética , Inflamação/metabolismo , Interleucinas/imunologia , Interleucinas/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Imunológicos , Mutação , Proteólise , Interferência de RNA , Proteínas de Ligação a RNA , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17/deficiência , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/imunologia , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Transdução de Sinais/imunologia , Fator 4 Associado a Receptor de TNF/deficiência , Fator 4 Associado a Receptor de TNF/genética , Tirosina/genética , Tirosina/imunologia , Tirosina/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Many malignant cells release the NKG2D ligand ULBP2 from their cell surface to evade immunosurveillance by NK cells and CD8 T cells. Although the shedding mechanism remains unclear, various inhibitors of matrix metalloproteinases have been shown to efficiently block the release of soluble ULBP2. The clinical use of these inhibitors, however, is limited because of adverse side effects. Using high-throughput screening technique, we identified a specific inhibitor of phosphatase of regenerating liver 3 (PRL-3) that could reduce the level of soluble ULBP2 in the culture supernatant of various cancer cell lines. Inhibition or gene knockdown of PRL-3 did not reduce ULBP2 shedding, but rather suppressed posttranslational maturation of ULBP2, resulting in intracellular retention of immature ULBP2. We then found that ULBP2 was constitutively associated with heat shock protein HSP60. Complete maturation of ULBP2 required tyrosine phosphorylation of HSP60 which was mediated by PRL-3.
Assuntos
Chaperonina 60/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Proteínas de Neoplasias/imunologia , Proteínas Tirosina Fosfatases/imunologia , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Chaperonina 60/metabolismo , Dipeptídeos/imunologia , Dipeptídeos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/farmacologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Células HCT116 , Células HEK293 , Células HT29 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Inibidores de Metaloproteinases de Matriz/imunologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Microscopia Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Ligação Proteica/imunologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/imunologia , Tirosina/metabolismoRESUMO
IL-7 is critical for murine T and B cell development and survival and plays a significant role in lymphoblastic leukemia in both humans and mice. We evaluated the role of the IL-7Rα Tyr(449) cytoplasmic SH2-binding motif in IL-7-mediated B cell development using a knock-in mouse with a Tyr to Phe mutation (IL-7Rα(449F/449F) mouse). IL-7Rα(449F/449F) and IL-7Rα(-/-) mice showed no defect in the number of pre-pro-B cells, although IL-7Rα(449F/449F) mice had decreased Ebf1 in pre-pro-B cells and impairment in B cell-committed CLPs. We identified that IL-7Rα Tyr(449) was critical for both pro-B and pre-B stages of development in the bone marrow. IL-7Rα(449F/449F) and IL-7Rα(-/-) mice had comparable precursor B cell defects, indicating that signaling from the IL-7Rα required this motif. Although the defect in IL-7Rα(449F/449F) pro-B cells was associated with loss of STAT5 activation and diminished expression of Mcl1, this was not rescued by overexpression of Bcl-2. IL-7Rα(449F/449F) and IL-7Rα(-/-) pre-B cells also showed defective cyto-Igµ and CD25 expression, associated with reduced levels of Rag1, Rag2, and Irf4. Pre-B cells from IL-7Rα(449F/449F) mice also failed to proliferate, perhaps as a result of the failure to rearrange Igµ. Our data suggest that IL-7Rα Tyr(449) was essential for IL-7Rα signaling in bone marrow B cell development and survival.
Assuntos
Medula Óssea/imunologia , Diferenciação Celular/imunologia , Mutação de Sentido Incorreto , Células Precursoras de Linfócitos B/imunologia , Receptores de Interleucina-7/imunologia , Transdução de Sinais/imunologia , Substituição de Aminoácidos , Animais , Diferenciação Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito B/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Humanos , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/imunologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/imunologia , Células Precursoras de Linfócitos B/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Receptores de Interleucina-7/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais/genética , Tirosina/genética , Tirosina/imunologiaRESUMO
Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a major negative regulatory molecule for T-cell activation with a complex biology and function. CTLA-4 is known to regulate homeostatic lymphoproliferation as well as tolerance induction and has been proposed to be an important effector molecule by which Treg cells suppress immunity. The immunoregulatory properties of CTLA-4 are primarily mediated by competition with the costimulator CD28 for ligand binding but also by delivering negative signals to T cells through its cytoplasmic tail. In this study, we addressed the effect of directly mutating the amino acid residue, Tyrosine 201 (Tyr201), of the intracellular domain of CTLA-4 in situ and its implications in T-cell function in the context of autoimmunity. Therefore, a novel CTLA-4 knock-in mouse (Y201V KI) was generated, in which Tyr201 was replaced by a valine that could not be phosphorylated. Mice expressing the CTLA-4 mutant molecule were generally healthy and did not show signs of disruption of T-cell homeostasis under steady-state conditions seen in CTLA-4 deficient mice. However, T cells isolated from Y201V KI mice expressed higher levels of CTLA-4 on the cell surface and displayed a Th2-biased phenotype following TCR stimulation. Furthermore, Y201V KI mice developed exacerbated disease as compared to wild-type upon antigen-specific T-cell activation in an in vivo model of EAE. Importantly, the Y201V mutation resulted in impaired suppressive activity of Treg cells while T effector function remained intact. These data suggest that effects associated with and mediated through Tyr201 of CTLA-4s intracellular domain are critical for Treg-cell function.
Assuntos
Antígeno CTLA-4/imunologia , Tolerância Imunológica , Linfócitos T Reguladores/imunologia , Substituição de Aminoácidos , Animais , Antígeno CTLA-4/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Linfócitos T Reguladores/patologia , Tirosina/genética , Tirosina/imunologiaRESUMO
We have recently described a method for tyrosine-ligation of complex glycans that was proven efficient for the site selective coupling of GBS capsular polysaccharides (PSs). Herein, we explored the effect of conjugation of type V polysaccharide onto predetermined lysine or tyrosine residues of the GBS67 pilus protein with the dual role of T-cell carrier for the PS and antigen. For the preparation of a conjugate at predetermined lysine residues of the protein, we investigated a two-step procedure based on microbial Transglutaminase (mTGase) catalyzed insertion of a tag bearing an azide for following copper-free strain-promoted azide-alkyne [3 + 2] cycloaddition (SPAAC) with the polysaccharide. Two glycoconjugates were obtained by tyrosine-ligation through the known SPAAC and a novel thiol-maleimide addition based approach. Controls were prepared by random conjugation of PSV to GBS67 and CRM197, a carrier protein present in many commercial vaccines. Immunological evaluation in mice showed that all the site-directed constructs were able to induce good levels of anti-polysaccharide and anti-protein antibodies inducing osponophagocytic killing of strains expressing individually PSV or GBS67. GBS67 randomly conjugated to PSV showed carrier properties similar to CRM197. Among the tested site-directed conjugates, tyrosine-directed ligation and thiol-malemide addition was elected as the best combination to ensure production of anti-polysaccharide and anti-protein functional antibodies (in vitro opsonophagocytic killing titers) comparable to the controls made by random conjugation, while avoiding anti-linker antibodies. Our findings demonstrate that (i) mTGase based conjugation at lysine residues is an alternative approach for the synthesis of large capsular polysaccharide-protein conjugates; (ii) GBS67 can be used with the dual role of antigen and carrier for PSV; and (iii) thiol-maleimide addition in combination with tyrosine-ligation ensures the production of anti-polysaccharide and anti-protein functional antibodies while maintaining low levels of anti-linker antibodies. Site-specific conjugation methods aid in defining conjugation site and chemistry in carbohydrate-protein conjugates.