Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
Immunity ; 44(5): 1091-101, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27192576

RESUMO

Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRß transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.


Assuntos
Imunidade Adaptativa , Colesterol/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Regulação Alostérica , Antígenos/imunologia , Antígenos/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Modelos Imunológicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Transdução de Sinais
2.
EMBO Rep ; 22(11): e51696, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34569685

RESUMO

Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.


Assuntos
Astrócitos , Microglia , Animais , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microglia/metabolismo , Doenças Neuroinflamatórias
3.
Int Immunol ; 28(3): 127-37, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26507128

RESUMO

Modulation of TCR signaling upon ligand binding is achieved by changes in the equilibrium between TCR degradation, recycling and synthesis; surprisingly, the molecular mechanism of such an important process is not fully understood. Here, we describe the role of a new player in the mediation of TCR degradation: the endocytic adaptor Numb. Our data show that Numb inhibition leads to abnormal intracellular distribution and defective TCR degradation in mature T lymphocytes. In addition, we find that Numb simultaneously binds to both Cbl and a site within CD3ε that overlaps with the Nck binding site. As a result, Cbl couples specifically to the CD3ε chain to mediate TCR degradation. The present study unveils a novel role of Numb that lies at the heart of TCR signaling initiation and termination.


Assuntos
Complexo CD3/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Oncogênicas/metabolismo , Ligação Proteica/genética , Transporte Proteico/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Deleção de Sequência/genética
5.
J Immunol ; 192(5): 2042-53, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24470497

RESUMO

On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-rich sequence (PRS) in CD3ε. We have studied the relevance of this interaction for T cell activation in vitro and in vivo by targeting the interaction sites in both partners. The first approach consisted of studying a knockin (KI) mouse line (KI-PRS) bearing a conservative mutation in the PRS that makes the TCR incompetent to recruit Nck. This deficiency prevents T cell activation by Ag in vitro and inhibited very early TCR signaling events including the tyrosine phosphorylation of CD3ζ. Most important, KI-PRS mice are partly protected against the development of neurological symptoms in an experimental autoimmune encephalitis model, and show a deficient antitumoral response after vaccination. The second approach consisted of using a high-affinity peptide that specifically binds the src homology 3.1 domain and prevents the interaction of Nck with CD3ε. This peptide inhibits T cell proliferation in vitro and in vivo. These data suggest that Nck recruitment to the TCR is fundamental to mount an efficient T cell response in vivo, and that the Nck-CD3ε interaction may represent a target for pharmacological modulation of the immune response.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Complexo CD3/imunologia , Ativação Linfocitária/fisiologia , Proteínas Oncogênicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos/genética , Antígenos/imunologia , Complexo CD3/genética , Proliferação de Células , Humanos , Células Jurkat , Camundongos , Camundongos Mutantes , Proteínas Oncogênicas/genética , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/fisiologia , Linfócitos T/citologia
6.
J Immunol ; 190(3): 1103-12, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23267019

RESUMO

The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4(+)CD8(+) thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo CD3/genética , Linfopoese/fisiologia , Proteínas Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timócitos/metabolismo , Timo/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Apresentação de Antígeno , Complexo CD3/imunologia , Antígenos CD4/análise , Antígenos CD8/análise , Células COS , Chlorocebus aethiops , Ativação Enzimática , Feminino , Técnicas de Introdução de Genes , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Domínios Proteicos Ricos em Prolina/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Timócitos/citologia , Timo/crescimento & desenvolvimento , Domínios de Homologia de src
7.
Commun Biol ; 7(1): 918, 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-39080357

RESUMO

Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.


Assuntos
Quinases Lim , Fosfoproteínas Fosfatases , Receptores de Antígenos de Linfócitos T , Humanos , Quinases Lim/metabolismo , Quinases Lim/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genética , Actinas/metabolismo , Actinas/genética , Ativação Linfocitária , Células Jurkat , Linfócitos T/metabolismo , Linfócitos T/imunologia , Transdução de Sinais , Sinapses Imunológicas/metabolismo
8.
Front Immunol ; 13: 799919, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35432331

RESUMO

Tγδ17 cells have emerged as a key population in the development of inflammatory and autoimmune conditions such as psoriasis. Thus, the therapeutic intervention of Tγδ17 cells can exert protective effects in this type of pathologies. Tγδ cells commit to IL-17 production during thymus development, and upon immune challenge, additional extrathymic signals induce the differentiation of uncommitted Tγδ cells into Tγδ17 effector cells. Despite the interest in Tγδ17 cells during the past 20 years, the role of TCR signaling in the generation and function of Tγδ17 cells has not been completely elucidated. While some studies point to the notion that Tγδ17 differentiation requires weak or no TCR signaling, other works suggest that Tγδ17 require the participation of specific kinases and adaptor molecules downstream of the TCR. Here we have examined the differentiation and pathogenic function of Tγδ17 cells in "knockin" mice bearing conservative mutations in the CD3ε polyproline rich sequence (KI-PRS) with attenuated TCR signaling due to lack of binding of the essential adaptor Nck. KI-PRS mice presented decreased frequency and numbers of Tγδ17 cells in adult thymus and lymph nodes. In the Imiquimod model of skin inflammation, KI-PRS presented attenuated skin inflammation parameters compared to wild-type littermates. Moreover, the generation, expansion and effector function Tγδ17 cells were impaired in KI-PRS mice upon Imiquimod challenge. Thus, we conclude that an intact CD3ε-PRS sequence is required for optimal differentiation and pathogenic function of Tγδ17 cells. These data open new opportunities for therapeutic targeting of specific TCR downstream effectors for treatment of Tγδ17-mediated diseases.


Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Animais , Imiquimode , Inflamação/metabolismo , Camundongos , Mutação , Peptídeos
9.
Cell Rep ; 34(11): 108861, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33730591

RESUMO

T cells form immunological synapses with professional antigen-presenting cells (APCs) resulting in T cell activation and the acquisition of peptide antigen-MHC (pMHC) complexes from the plasma membrane of the APC. They thus become APCs themselves. We investigate the functional outcome of T-T cell antigen presentation by CD4 T cells and find that the antigen-presenting T cells (Tpres) predominantly differentiate into regulatory T cells (Treg), whereas T cells that have been stimulated by Tpres cells predominantly differentiate into Th17 pro-inflammatory cells. Using mice deficient in pMHC uptake by T cells, we show that T-T antigen presentation is important for the development of experimental autoimmune encephalitis and Th17 cell differentiation in vivo. By varying the professional APC:T cell ratio, we can modulate Treg versus Th17 differentiation in vitro and in vivo, suggesting that T-T antigen presentation underlies proinflammatory responses in conditions of antigen scarcity.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos/metabolismo , Polaridade Celular/imunologia , Células Th17/imunologia , Animais , Antígenos CD28/metabolismo , Diferenciação Celular/imunologia , Membrana Celular/metabolismo , Células Dendríticas/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica , Genoma , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Transcrição Gênica , Trogocitose , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/metabolismo
10.
Biochim Biophys Acta ; 1794(1): 110-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18955169

RESUMO

The first SH3 domain (SH3.1) of Nckalpha specifically recognizes the proline-rich region of CD3varepsilon, a subunit of the T cell receptor complex. We have solved the NMR structure of Nckalpha SH3.1 that shows the characteristic SH3 fold consisting of two antiparallel beta-sheets tightly packed against each other. According to chemical shift mapping analysis, a peptide encompassing residues 150-166 of CD3varepsilon binds at the canonical SH3 binding site. An exhaustive comparison with the structures of other SH3 domains able and unable to bind CD3varepsilon reveals that Nckalpha SH3.1 recognises a non-canonical PxxPxxDY motif that orientates at the binding site as a class II ligand. A positively charged residue (K/R) at position -2 relative to the WW sequence at the beginning of strand beta3 is crucial for PxxDY recognition. A 14-mer optimised Nckalpha SH3.1 ligand was found using a multi-substitution approach. Based on NMR data, this improved ligand binds Nckalpha SH3.1 through a PxxPxRDY motif that combines specific stabilising interactions corresponding to both canonical class II, PxxPx(K/R), and non-canonical PxxPxxDY motifs. This explains its higher capacity for Nckalpha SH3.1 binding relative to the wild type sequence.


Assuntos
Complexo CD3/química , Proteínas Oncogênicas/química , Peptídeos/química , Peptídeos/metabolismo , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Ligantes , Ativação Linfocitária , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Prolina/química , Prolina/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo
11.
Life Sci Alliance ; 1(5): e201800060, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30456377

RESUMO

During B-cell activation, the dynamic reorganisation of the cytoskeleton is crucial for multiple cellular responses, such as receptor signalling, cell spreading, antigen internalisation, intracellular trafficking, and antigen presentation. However, the role of intermediate filaments (IFs), which represent a major component of the mammalian cytoskeleton, is not well defined. Here, by using multiple super-resolution microscopy techniques, including direct stochastic optical reconstruction microscopy, we show that IFs in B cells undergo drastic reorganisation immediately upon antigen stimulation and that this reorganisation requires actin and microtubules. Although the loss of vimentin in B cells did not impair B-cell development, receptor signalling, and differentiation, vimentin-deficient B cells exhibit altered positioning of antigen-containing and lysosomal associated membrane protein 1 (LAMP1+) compartments, implying that vimentin may play a role in the fine-tuning of intracellular trafficking. Indeed, vimentin-deficient B cells exhibit impaired antigen presentation and delayed antibody responses in vivo. Thus, our study presents a new perspective on the role of IFs in B-cell activation.

12.
Nat Commun ; 9(1): 1080, 2018 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540686

RESUMO

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P212 in human vs. A210 in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this Y209APP212 sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside.


Assuntos
Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/fisiologia , Camundongos , NF-kappa B/metabolismo , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Transdução de Sinais/genética , Linfócitos T Reguladores/metabolismo
13.
Nat Commun ; 9(1): 495, 2018 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-29386506

RESUMO

The original version of this Article contained an error in the spelling of the author José María González-Granado, which was incorrectly given as José María Gozález-Granado. This has now been corrected in both the PDF and HTML versions of the Article.

14.
Nat Commun ; 8(1): 1591, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-29147022

RESUMO

Bacterial phagocytosis and antigen cross-presentation to activate CD8+ T cells are principal functions of professional antigen presenting cells. However, conventional CD4+ T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8+ T cells, which proliferate and become cytotoxic in response. CD4+ T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8+ T cells with low PD-1 expression. Moreover, transphagocytic CD4+ T cells induce protective anti-tumour immune responses by priming CD8+ T cells, highlighting the potential of CD4+ T cells as a tool for cancer immunotherapy.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Animais , Células Cultivadas , Apresentação Cruzada/imunologia , Citotoxicidade Imunológica/imunologia , Memória Imunológica/imunologia , Sinapses Imunológicas/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/imunologia , Receptor de Morte Celular Programada 1/imunologia
15.
Nat Commun ; 7: 11389, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27091106

RESUMO

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.


Assuntos
Aurora Quinase A/genética , Vesículas Citoplasmáticas/imunologia , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/imunologia , Azepinas/farmacologia , Complexo CD3/genética , Complexo CD3/imunologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/ultraestrutura , Feminino , Regulação da Expressão Gênica , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/genética , Interleucina-2/genética , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Ativação Linfocitária/efeitos dos fármacos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Microtúbulos/efeitos dos fármacos , Microtúbulos/imunologia , Microtúbulos/ultraestrutura , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/ultraestrutura
16.
Sci Transl Med ; 8(370): 370ra184, 2016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-28003549

RESUMO

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Administração Oral , Animais , Anti-Inflamatórios/farmacologia , Doenças Autoimunes/imunologia , Proliferação de Células , Citocinas/metabolismo , Desenho de Fármacos , Feminino , Voluntários Saudáveis , Humanos , Terapia de Imunossupressão , Concentração Inibidora 50 , Ligantes , Ativação Linfocitária , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Linfócitos T/citologia
17.
Immunol Lett ; 161(1): 113-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24877875

RESUMO

Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering.


Assuntos
Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Humanos , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química
18.
Sci Signal ; 7(354): ra115, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25468995

RESUMO

In the thymus, immature T cells differentiate from common precursors to become T cells expressing either the αß or γδ T cell receptor (TCR) complex. The CD3ε subunit of the TCR complex is thought to transduce ligand-induced conformational changes in the TCR by recruiting the cytosolic adaptor protein Nck. To investigate the role of conformational changes in the TCR in T cell development, we generated mice with a germline mutation (C80G) in the extracellular domain of CD3ε, which prevents the outside-in transmission of conformational changes in the TCR. The development of αß T cells in the C80G mice was blocked at an early stage that depends on signaling by a precursor form of the TCR. In contrast, the C80G mutation did not impair the development of some subsets of γδ T cells, including Vγ1.1(+) cells; however, development of other γδ T cell subsets was blocked. A similar phenotype was observed in mice with a mutation in the cytoplasmic proline-rich sequence (PRS) of CD3ε, the binding site for Nck. In a genetic complementation test, the PRS CD3ε mutant failed to rescue the wild-type phenotype when expressed in heterozygosity with the C80G mutant. These data suggest that Nck may function as an effector of TCR conformational changes during T cell development. Additional experiments showed differential effects of the C80G mutation on the activation of TCR-dependent signaling pathways, which suggests that there are pathways that are either dependent on or independent of the transmission of conformational change in the receptor.


Assuntos
Diferenciação Celular/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Complexo CD3/genética , Complexo CD3/metabolismo , Citometria de Fluxo , Técnicas de Introdução de Genes , Teste de Complementação Genética , Mutação em Linhagem Germinativa/genética , Imunoprecipitação , Camundongos , Proteínas Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética
19.
J Biol Chem ; 280(44): 36970-7, 2005 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-16129682

RESUMO

Impairments in signal transduction, leading to the regulation of cell proliferation, differentiation, or migration are frequently the cause of cancer. Since the accurate spatial and temporal location of their components is crucial to ensure the correct regulation of these signaling pathways, it could be anticipated that defects in intracellular trafficking are at the base of certain neoplasias. However, the trafficking of many components of pathways frequently up-regulated in cancers, such as the epidermal growth factor receptor (EGFR) pathway, are largely unknown. Here, we show that the pro-transforming growth factor-alpha (pro-TGF-alpha), a prototypical EGFR ligand, is endocytosed from the cell surface via a clathrin-dependent pathway. Internalized pro-TGF-alpha does not progress to the lysosome; instead, it is delivered to the cell surface via recycling endosomes. To analyze the functional meaning of the internalization of pro-TGF-alpha, we used a deletion construct that is normally transported to the cell surface but is deficiently endocytosed. Due to this impairment, the levels of this construct at the cell surface are dramatically augmented. Consequently, the deletion construct displays a higher EGFR-activating ability, revealing a link between the trafficking of pro-TGF-alpha and the signaling by the EGFR and opening the possibility that defects in the trafficking of the growth factor may contribute to the development of tumors.


Assuntos
Membrana Celular/metabolismo , Endocitose/fisiologia , Receptores ErbB/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador alfa/metabolismo , Animais , Células CHO , Proliferação de Células , Clatrina/metabolismo , Cricetinae , Cães , Endossomos/metabolismo , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Lisossomos/metabolismo , Deleção de Sequência
20.
J Biol Chem ; 278(28): 25933-9, 2003 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-12714588

RESUMO

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.


Assuntos
Metaloendopeptidases/metabolismo , Mutação , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animais , Biotinilação , Western Blotting , Células CHO , Linhagem Celular , Cricetinae , DNA Complementar/metabolismo , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Glicoproteínas/química , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Testes de Precipitina , Estrutura Terciária de Proteína , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator de Crescimento Transformador alfa/metabolismo , Células Tumorais Cultivadas , Zinco/metabolismo , alfa 1-Antitripsina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA