RESUMO
Assembly of a scaffold consisting of CARD9, BCL10, and MALT1 (CBM complex) is critical for effective signaling by multiple pattern recognition receptors (PRRs) including Dectin and RIG-I. The RUN domain Beclin-1-interacting cysteine-rich-containing Rubicon protein associates constitutively with the Beclin-UVRAG-Vps34 complex under normal conditions to regulate autophagy. Rubicon also interacts with the phagocytic NADPH-oxidase complex upon TLR stimulation to induce potent antimicrobial responses. Here, we show Rubicon is a physiological feedback inhibitor of CBM-mediated PRR signaling, preventing unbalanced proinflammatory responses. Upon Dectin-1- or RIG-I-mediated activation, Rubicon dynamically exchanges binding partners from 14-3-3ß to CARD9 in a stimulation-specific and phosphorylation-dependent manner, disassembling the CBM signaling complex and ultimately terminating PRR-induced cytokine production. Remarkably, Rubicon's actions in the autophagy complex, phagocytosis complex, and CBM complex are functionally and genetically separable. Rubicon thus differentially targets signaling complexes, depending on environmental stimuli, and may function to coordinate various immune responses against microbial infection.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Proteína 10 de Linfoma CCL de Células B , Ligação Competitiva , Candida albicans/crescimento & desenvolvimento , Candida albicans/imunologia , Candida albicans/fisiologia , Candidíase/imunologia , Candidíase/metabolismo , Caspases/metabolismo , Células Cultivadas , Citocinas/biossíntese , Retroalimentação Fisiológica , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Transgênicos , Viabilidade Microbiana , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de SinaisRESUMO
The C-type lectin receptors (CLRs) belong to a large family of proteins that contain a carbohydrate recognition domain (CRD) and calcium binding sites on their extracellular domains. Recent studies indicate that many CLRs, such as Dectin-1, Dectin-2 and Mincle, function as pattern recognition receptors (PRRs) recognizing carbohydrate ligands from infected microorganisms. Upon ligand binding, these CLRs induce multiple signal transduction cascades through their own immunoreceptor tyrosine-based activation motifs (ITAMs) or interacting with ITAM-containing adaptor proteins such as FcRγ. Emerging evidence indicate that CLR-induced signaling cascades lead to the activation of nuclear factor kappaB (NF-κB) family of transcriptional factors through a Syk- and CARD9-dependent pathway(s). The activation of NF-κB plays a critical role in the induction of innate immune and inflammatory responses following microbial infection and tissue damages. In this review, we will summarize the recent progress on the signal transduction pathways induced by CLRs, and how these CLRs activate NF-κB and contribute to innate immune and inflammatory responses.
Assuntos
Inflamação/metabolismo , Lectinas Tipo C/metabolismo , NF-kappa B/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Humanos , Imunidade Inata/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/imunologia , NF-kappa B/genética , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais/imunologia , Quinase Syk , Ativação Transcricional/imunologiaRESUMO
The immune response to the tumor can be enhanced by targeting costimulatory molecules on T cells. As the CD70-CD27 costimulatory axis plays an important role in the activation, survival, and differentiation of lymphocytes, we have examined the efficacy of agonistic anti-CD27 antibodies as monotherapies for established melanoma in a murine model. We show that this approach leads to a substantial reduction in the outgrowth of both experimental lung metastases and subcutaneous tumors. Anti-CD27 treatment supports the maintenance of tumor-specific CD8(+) T cells within the tumor, reduces the frequency of FoxP3-expressing CD4(+) T cells within tumors, and potentiates the ability of NK1.1(+) and CD8(+) tumor infiltrating cells to secrete IFNγ upon coculture with tumor cells. The enhanced effector function correlated with lower levels of PD-1 expression on CD8(+) T cells from anti-CD27-treated mice. Despite the modulating effect of anti-CD27 on multiple cell types, only CD8(+) T cells were absolutely required for tumor control. The CD4(+) T cells were dispensable, whereas NK1.1(+) cells were needed during early stages of tumor growth but not for the effectiveness of anti-CD27. Thus, CD27-mediated costimulation provides a potent boost to multiple aspects of the endogenous responses to tumor, and may be exploited to enhance tumor immunity.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Imunoterapia , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Transcrição Forkhead/biossíntese , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Melanoma Experimental/secundário , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1 , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Carga Tumoral/efeitos dos fármacos , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
TCR-mediated activation of the transcription factor NF-κB is required for T cell proliferation, survival, and effector differentiation. Although this pathway is the subject of intense study, it is not known whether TCR signaling to NF-κB is digital (switch-like) or analog in nature. Through analysis of the phosphorylation and degradation of IκBα and the nuclear translocation and phosphorylation of the NF-κB subunit RelA, we show that TCR-directed NF-κB activation is digital. Furthermore, digitization occurs well upstream of the IκB kinase complex, as protein kinase C translocation to the immunologic synapse and activation-associated aggregation of Bcl10 and Malt1 also demonstrate both digital behavior and high correlation with RelA nuclear translocation. Thus, similar to the TCR-to-MAPK signaling cascade, analog Ag inputs are converted to digital activation outputs to NF-κB at an early step downstream of TCR ligation.
Assuntos
Ativação Linfocitária/imunologia , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Animais , Separação Celular , Ativação Enzimática , Citometria de Fluxo , Humanos , Proteínas I-kappa B/imunologia , Proteínas I-kappa B/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa , NF-kappa B/imunologia , Fosforilação , Transporte Proteico/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Malt1 is a multi-domain cytosolic signaling molecule that was originally identified as the target of recurrent translocations in a large fraction of MALT lymphomas. The product of this translocation is a chimeric protein in which the N-terminus is contributed by the apoptosis inhibitor, cIAP2, and the C-terminus is contributed by Malt1. Early studies suggested that Malt1 is an essential intermediate in antigen receptor activation of NF-kappaB, and that the juxtaposition of the cIAP2 N-terminus and the Malt1 C-terminus results in deregulation of Malt1 NF-kappaB stimulatory activity. Initial experimental data further suggested that the molecular mechanisms of Malt1- and cIAP-Malt1-mediated NF-kappaB activation were quite similar. However, a number of more recent studies of both Malt1 and cIAP2-Malt1 now reveal that these proteins influence NF-kappaB activation by multiple distinct mechanisms, several of which are non-overlapping. Currently available data suggest a revised model in which cIAP2-Malt1 induces NF-kappaB activation via a mechanism that depends equally on domains contributed by cIAP2 and Malt1, which confer spontaneous oligomerization activity, polyubiquitin binding, proteolytic activity, and association with and activation of TRAF2 and TRAF6 at several independent binding sites. By contrast, emerging data suggest that the wild-type Malt1 protein uniquely contributes to NF-kappaB activation primarily through the control of two proteolytic cleavage mechanisms. Firstly, Malt1 directly cleaves and inactivates A20, a negative regulator of the antigen receptor-to-NF-kappaB pathway. Secondly, Malt1 interacts with caspase-8, inducing caspase-8 cleavage of c-FLIP(L), initiating a pathway that contributes to activation of the I kappaB kinase (IKK) complex. Furthermore, data suggest that Malt1 plays a more limited and focused role in antigen receptor activation of NF-kappaB, serving to augment weak antigen signals and stimulate a defined subset of NF-kappaB dependent responses. Thus, the potent activation of NF-kappaB by cIAP2-Malt1 contrasts with the more subtle role of Malt1 in regulating specific NF-kappaB responses downstream of antigen receptor ligation.
Assuntos
Caspases/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Humanos , Interleucina-2/biossíntese , Proteínas Proto-Oncogênicas c-rel/metabolismoRESUMO
BACKGROUND: Confocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level. Most basic confocal microscopes are equipped to cleanly discriminate no more than four fluorophores in a given sample, limiting the utility of this method for co-localization, co-expression, and other multi-parameter analyses. In this study, we evaluated the use of red and near-infrared emitting quantum dot staining reagents to expand the multi-parameter capabilities of basic confocal microscopes. RESULTS: We modified a three-laser Zeiss Pascal confocal microscope by the addition of two band-pass filters and one long-pass filter for the detection of three different red to near-infrared quantum dot conjugates. We then performed direct comparisons between organic dye- and quantum dot-labeled detection reagents for the detection of subcellular structures. We found that the quality of staining was generally indistinguishable, although quantum dot reagents do have certain limitations, relative to organic dye conjugates. Using the modified Pascal system, three quantum dot conjugates, two organic dye conjugates, and one fluorescent protein, we demonstrated clean discrimination of six distinct fluorescent labels in a single sample. CONCLUSION: Our data demonstrate that nearly any basic confocal microscope can be modified by the simple addition of appropriate emission filters, allowing the detection of red and near-infrared quantum dot conjugates. Additionally, quantum dot- and organic dye-based secondary reagents can be successfully combined in complex intracellular staining experiments. Substantial expansion of the multi-parameter capabilities of basic confocal instruments can be achieved with a financial investment that is minimal in comparison to instrument replacement or upgrade with additional lasers.
Assuntos
Corantes Fluorescentes/química , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Pontos Quânticos , Animais , Camundongos , Células NIH 3T3RESUMO
The cytosolic proteins protein kinase Ctheta (PKCtheta), Bcl10, and Malt1 play critical roles in TCR signaling to the transcription factor NF-kappaB. Our data confirm that CD4(+) T cells from PKCtheta, Bcl10, and Malt1 knockout mice show severe impairment of proliferation in response to TCR stimulation. Unexpectedly, we find that knockout CD8(+) T cells proliferate to a similar extent as wild-type cells in response to strong TCR signals, although a survival defect prevents their accumulation. Both CD4(+) and CD8(+) knockout T cells express activation markers, including CD25, following TCR stimulation. Addition of exogenous IL-2 rescues survival of knockout CD4(+) and CD8(+) T cells, but fails to overcome the proliferation defect of CD4(+) T cells. CD4(+) T cells from knockout mice are extremely deficient in TCR-induced NF-kappaB activation, whereas NF-kappaB activation is only partially impaired in CD8(+) T cells. Overall, our results suggest that defects in TCR signaling through PKCtheta, Bcl10, and Malt1 predominantly impair NF-kappaB activation and downstream functional responses of CD4(+) T cells. In contrast, CD8(+) T cells maintain substantial NF-kappaB signaling, implying the existence of a significant TCR-regulated NF-kappaB activation pathway in CD8(+) T cells that is independent of PKCtheta, Bcl10, and Malt1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Caspases/deficiência , Proliferação de Células , Regulação para Baixo/imunologia , Isoenzimas/deficiência , NF-kappa B/antagonistas & inibidores , Proteínas de Neoplasias/deficiência , Proteína Quinase C/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Animais , Proteína 10 de Linfoma CCL de Células B , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Caspases/genética , Caspases/fisiologia , Células Cultivadas , Regulação para Baixo/genética , Isoenzimas/genética , Isoenzimas/fisiologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitomicina/imunologia , Dados de Sequência Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Ovalbumina/imunologia , Proteína Quinase C/genética , Proteína Quinase C/fisiologia , Proteína Quinase C-theta , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Bcl10 and MALT1 are essential mediators of NF-kappaB activation in response to the triggering of a diverse array of transmembrane receptors, including antigen receptors. Additionally, both proteins are translocation targets in MALT lymphoma. Thus, a detailed understanding of the interaction between these mediators is of considerable biological importance. Previous studies have indicated that a 13-amino acid region downstream of the Bcl10 caspase recruitment domain (CARD) is responsible for interacting with the immunoglobulin-like domains of MALT1. We now provide evidence that the death domain of MALT1 and the CARD of Bcl10 also contribute to Bcl10-MALT1 interactions. Although a direct interaction between the MALT1 death domain and Bcl10 cannot be detected via immunoprecipitation, FRET data strongly suggest that the death domain of MALT1 contributes significantly to the association between Bcl10 and MALT1 in T cells in vivo. Furthermore, analysis of point mutants of conserved residues of Bcl10 shows that the Bcl10 CARD is essential for interaction with the MALT1 N terminus. Mutations that disrupt proper folding of the Bcl10 CARD strongly impair Bcl10-MALT1 interactions. Molecular modeling and functional analyses of Bcl10 point mutants suggest that residues Asp(80) and Glu(84) of helix 5 of the Bcl10 CARD directly contact MALT1. Together, these data demonstrate that the association between Bcl10 and MALT1 involves a complex interaction between multiple protein domains. Moreover, the Bcl10-MALT1 interaction is the second reported example of interactions between a CARD and a non-CARD protein region, which suggests that many signaling cascades may utilize CARD interactions with non-CARD domains.