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1.
J Exp Med ; 219(8)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35766979

RESUMO

Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (Ptsn), a regulatory subunit of protein phosphatase 1, is a component of the complex. Ptsn mediates dephosphorylation of Rap1, thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes Ptsn, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable, in part, to defective activation of integrins αLß2 and α4ß7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, Ptsn enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of Ptsn ameliorates T cell-mediated colitis.


Assuntos
Integrinas , Tecido Linfoide , Proteína Fosfatase 1 , Linfócitos T , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Adesão Celular/fisiologia , Colite/imunologia , Colite/metabolismo , Integrinas/imunologia , Integrinas/metabolismo , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteína Fosfatase 1/imunologia , Proteína Fosfatase 1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Talina/metabolismo , Proteínas rap1 de Ligação ao GTP/imunologia , Proteínas rap1 de Ligação ao GTP/metabolismo
2.
Toxins (Basel) ; 13(11)2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34822549

RESUMO

Cyanobacteria (blue-green algae) have been present on Earth for over 2 billion years, and can produce a variety of bioactive molecules, such as cyanotoxins. Microcystins (MCs), the most frequently detected cyanotoxins, pose a threat to the aquatic environment and to human health. The classic toxic mechanism of MCs is the inhibition of the protein phosphatases 1 and 2A (PP1 and PP2A). Immunity is known as one of the most important physiological functions in the neuroendocrine-immune network to prevent infections and maintain internal homoeostasis in fish. The present review aimed to summarize existing papers, elaborate on the MC-induced immunotoxicity in fish, and put forward some suggestions for future research. The immunomodulatory effects of MCs in fish depend on the exposure concentrations, doses, time, and routes of exposure. Previous field and laboratory studies provided strong evidence of the associations between MC-induced immunotoxicity and fish death. In our review, we summarized that the immunotoxicity of MCs is primarily characterized by the inhibition of PP1 and PP2A, oxidative stress, immune cell damage, and inflammation, as well as apoptosis. The advances in fish immunoreaction upon encountering MCs will benefit the monitoring and prediction of fish health, helping to achieve an ecotoxicological goal and to ensure the sustainability of species. Future studies concerning MC-induced immunotoxicity should focus on adaptive immunity, the hormesis phenomenon and the synergistic effects of aquatic microbial pathogens.


Assuntos
Apoptose/efeitos dos fármacos , Peixes , Imunotoxinas/toxicidade , Inflamação/imunologia , Microcistinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Animais , Doenças dos Peixes/induzido quimicamente , Doenças dos Peixes/imunologia , Peixes/imunologia , Peixes/metabolismo , Inflamação/induzido quimicamente , Proteína Fosfatase 1/imunologia , Proteína Fosfatase 2/imunologia
3.
Nat Microbiol ; 6(8): 1031-1042, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34282309

RESUMO

The antiviral cytokine interferon activates expression of interferon-stimulated genes to establish an antiviral state. Myxovirus resistance 2 (MX2, also known as MxB) is an interferon-stimulated gene that inhibits the nuclear import of HIV-1 and interacts with the viral capsid and cellular nuclear transport machinery. Here, we identified the myosin light chain phosphatase (MLCP) subunits myosin phosphatase target subunit 1 (MYPT1) and protein phosphatase 1 catalytic subunit-ß (PPP1CB) as positively-acting regulators of MX2, interacting with its amino-terminal domain. We demonstrated that serine phosphorylation of the N-terminal domain at positions 14, 17 and 18 suppresses MX2 antiviral function, prevents interactions with the HIV-1 capsid and nuclear transport factors, and is reversed by MLCP. Notably, serine phosphorylation of the N-terminal domain also impedes MX2-mediated inhibition of nuclear import of cellular karyophilic cargo. We also found that interferon treatment reduces levels of phosphorylation at these serine residues and outline a homeostatic regulatory mechanism in which repression of MX2 by phosphorylation, together with MLCP-mediated dephosphorylation, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Imunidade Inata , Proteínas de Resistência a Myxovirus/química , Proteínas de Resistência a Myxovirus/imunologia , Motivos de Aminoácidos , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Células HeLa , Humanos , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosfatase de Miosina-de-Cadeia-Leve/imunologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Proteínas de Resistência a Myxovirus/genética , Fosforilação , Domínios Proteicos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/imunologia , Serina/metabolismo
4.
Front Immunol ; 11: 609890, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33584687

RESUMO

Protein phosphatase-1 (PP1) has an important role in many cell functions, such as cell differentiation, development, immune response and tumorigenesis. However, the specific role of PP1 in the antiviral response in fish remains to be elucidated. In this study, the PPP1R3G homolog was identified in the grass carp (Ctenopharyngodon idella) and its role in defence against the GCRV infection was investigated. Phylogenetic analysis demonstrated that CiPPP1R3G clustered with homologues from other teleosts. Temporal expression analysis in vivo revealed that the expression level of CiPPP1R3G was significantly up-regulated in response to GCRV infection in grass carps, especially in the intestine and head-kidney. Cellular distribution analysis revealed that CiPPP1R3G was located in the nucleus and cytoplasm. Overexpression of CiPPP1R3G significantly negatively regulated the expression of CiIRF3, thus inhibiting its activation. In summary, we systematically analyzed the PPP1R3G gene in grass carp and illustrated its function as a negative regulator in the anti-GCRV immune responses.


Assuntos
Carpas/metabolismo , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Proteína Fosfatase 1/metabolismo , Infecções por Reoviridae/metabolismo , Sequência de Aminoácidos , Animais , Carpas/imunologia , Carpas/virologia , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/imunologia , Citoplasma/metabolismo , Doenças dos Peixes/metabolismo , Doenças dos Peixes/virologia , Proteínas de Peixes/imunologia , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/imunologia , Filogenia , Proteína Fosfatase 1/imunologia , Reoviridae/imunologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/virologia , Regulação para Cima/imunologia , Regulação para Cima/fisiologia
5.
J Clin Invest ; 129(7): 2717-2729, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31180338

RESUMO

Invasive fungal infection is a serious health threat with high morbidity and mortality. Current antifungal drugs only demonstrate partial success in improving prognosis. Furthermore, mechanisms regulating host defense against fungal pathogens remain elusive. Here, we report that the downstream of kinase 3 (Dok3) adaptor negatively regulates antifungal immunity in neutrophils. Our data revealed that Dok3 deficiency increased phagocytosis, proinflammatory cytokine production, and netosis in neutrophils, thereby enhancing mutant mouse survival against systemic infection with a lethal dose of the pathogenic fungus Candida albicans. Biochemically, Dok3 recruited protein phosphatase 1 (PP1) to dephosphorylate Card9, an essential player in innate antifungal defense, to dampen downstream NF-κB and JNK activation and immune responses. Thus, Dok3 suppresses Card9 signaling, and disrupting Dok3-Card9 interaction or inhibiting PP1 activity represents therapeutic opportunities to develop drugs to combat candidaemia.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Candida albicans/imunologia , Candidíase/imunologia , Neutrófilos/imunologia , Proteína Fosfatase 1/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Candidíase/genética , Candidíase/patologia , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Proteína Fosfatase 1/genética , Transdução de Sinais/genética
6.
Anticancer Res ; 37(12): 6705-6714, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29187447

RESUMO

BACKGROUND/AIM: Bladder cancer (BC) has a high recurrence rate and may progress to being a muscle-invasive lesion, that is potentially associated with a poor prognosis. We identified tumor-associated proteins that were recognized by autoantibodies in sera from patients with high-grade non-muscle-invasive bladder cancer (HG-NMIBC) by proteomic analysis. MATERIALS AND METHODS: The serum levels of these autoantibodies against identified proteins were validated by dot blot analysis with sera from 95 patients with BC and 35 healthy controls. The expression of identified proteins was immunohistochemically analyzed in 115 BC tissues. RESULTS: Autoantibody against protein phosphatase 1, catalytic subunit, alpha isoform (PPP1CA) protein was detected in pretreated sera from patients with HG-NMIBC who showed progression. The serum IgG level of anti-PPP1CA autoantibody was significantly correlated with pathological stage, grade, lymphovascular invasion, and prognosis. The immunoreactions for PPP1CA protein in BC was significantly correlated with pathological stage, grade, and lymphovascular invasion. CONCLUSION: PPP1CA is a candidate sero-diagnostic and prognostic marker for patients with BC.


Assuntos
Autoanticorpos/imunologia , Proteoma/imunologia , Proteômica/métodos , Neoplasias da Bexiga Urinária/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoanticorpos/sangue , Progressão da Doença , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Proteína Fosfatase 1/imunologia , Proteína Fosfatase 1/metabolismo , Neoplasias da Bexiga Urinária/sangue , Neoplasias da Bexiga Urinária/metabolismo
7.
Cell Death Dis ; 7: e2219, 2016 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-27171261

RESUMO

Growth arrest and DNA damage inducible protein 34 (GADD34) is induced by various cellular stresses, such as DNA damage, endoplasmic reticulum stress, and amino-acid deprivation. Although the major roles of GADD34 are regulating ER stress responses and apoptosis, a recent study suggested that GADD34 is linked to innate immune responses. In this report, we investigated the roles of GADD34 in inflammatory responses against bacterial infection. To explore the effects of GADD34 on systemic inflammation in vivo, we employed a lipopolysaccharide (LPS)-induced murine sepsis model and assessed the lethality, serum cytokine levels, and tissue injury in the presence or absence of GADD34. We found that GADD34 deficiency increased the lethality and serum cytokine levels in LPS-induced sepsis. Moreover, GADD34 deficiency enhanced tissue destruction, cell death, and pro-inflammatory cytokine expression in LPS-induced acute liver injury. Pro-inflammatory cytokine production after LPS stimulation is regulated by the Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway. In vitro experiments revealed that GADD34 suppressed pro-inflammatory cytokine production by macrophages through dephosphorylation of IKKß. In conclusion, GADD34 attenuates LPS-induced sepsis and acute tissue injury through suppressing macrophage activation. Targeting this anti-inflammatory role of GADD34 may be a promising area for the development of therapeutic agents to regulate inflammatory disorders.


Assuntos
Lesão Pulmonar Aguda/imunologia , Macrófagos Peritoneais/imunologia , NF-kappa B/imunologia , Proteína Fosfatase 1/imunologia , Sepse/imunologia , Receptor 4 Toll-Like/imunologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Regulação da Expressão Gênica , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Lipopolissacarídeos , Ativação de Macrófagos , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Cultura Primária de Células , Proteína Fosfatase 1/genética , Sepse/induzido quimicamente , Sepse/genética , Sepse/patologia , Transdução de Sinais , Receptor 4 Toll-Like/genética
8.
J Immunol ; 192(7): 3143-55, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24574500

RESUMO

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.


Assuntos
Monócitos/imunologia , NF-kappa B/imunologia , Proteína Fosfatase 1/imunologia , Transdução de Sinais/imunologia , Fator de Transcrição AP-1/imunologia , Fator de Necrose Tumoral alfa/imunologia , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Tolerância a Medicamentos/imunologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/imunologia , Quinase 3 da Glicogênio Sintase/metabolismo , Células HeLa , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Fator de Necrose Tumoral alfa/farmacologia
9.
J Immunol ; 192(6): 2846-56, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24534530

RESUMO

The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-κB pathway and the production of TNF-α, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-ß-activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R-triggered NF-κB and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-κB and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses.


Assuntos
MAP Quinase Quinase Quinases/imunologia , Proteína Fosfatase 1/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Animais , Linhagem Celular , Células Cultivadas , Furanos/farmacologia , Células HEK293 , Células HeLa , Holoenzimas/genética , Holoenzimas/imunologia , Holoenzimas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Lipídeos/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Imunológicos , Mutação/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosforilação/imunologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Interferência de RNA , Serina/genética , Serina/imunologia , Serina/metabolismo , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
11.
Immunol Cell Biol ; 92(2): 170-80, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24247289

RESUMO

Hematopoietic stem cells (HSCs) generate all known hematopoietic lineages and are capable of self-renewal. Upon aging, myeloid-biased HSCs are maintained, whereas lymphoid-biased HSCs are lost. GADD34 protein is expressed in myeloid-lineage cells and has been cloned from them. However, the function of GADD34 in the myeloid lineage has not yet been elucidated. Here, we show that early age-dependent deviation to the myeloid lineage occurs in GADD34-deficient mice. Early increases of GR-1(int)CD11b(+) and GR-1(high)CD11b(+) neutrophils were observed in the spleen, bone marrow (BM) and blood of GADD34-deficient mice. We found that BM Lin(-) c-Kit(+) Sca1(+) and Lin(-) c-Kit(+) Sca1(-)cells expressed GADD34 protein without stimulation and increased GADD34 expression following intravenous injection of Staphylococcus aureus (S.aureus). These cell populations were high in GADD34-deficient BM and were increased by the injection of S. aureus. Because of the increase in granulocyte colony-stimulating factor (G-CSF) induced by S. aureus injection, we examined the signaling pathway from the G-CSF receptor (G-CSFR). We found that phosphorylation of signal transducer and activator of transcription factor 3 was highly increased in GADD34-deficient Lin(-) BM cells by the stimulation of G-CSF. These results indicate that GADD34 binds to Lyn and inhibit G-CSFR signaling. We show here that GADD34 works to inhibit the proliferation and differentiation of HSCs or myeloid precursor cells and maintains homeostatic differentiation of neutrophil-lineage cells to avoid early immunological senescence.


Assuntos
Envelhecimento/imunologia , Diferenciação Celular/imunologia , Células Progenitoras Mieloides/imunologia , Proteína Fosfatase 1/imunologia , Transdução de Sinais/imunologia , Quinases da Família src/imunologia , Envelhecimento/genética , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Diferenciação Celular/genética , Fator Estimulador de Colônias de Granulócitos/imunologia , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Fosforilação/genética , Fosforilação/imunologia , Proteína Fosfatase 1/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/imunologia , Transdução de Sinais/genética , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/imunologia , Quinases da Família src/genética
12.
J Immunol ; 190(12): 6034-42, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23690473

RESUMO

Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.


Assuntos
Epitopos Imunodominantes/imunologia , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Fosfoproteínas Fosfatases/imunologia , Proteína Fosfatase 1/imunologia , Sequência de Bases , Ensaios Clínicos como Assunto , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Humanos , Ativação Linfocitária/imunologia , Masculino , Melanoma/genética , Dados de Sequência Molecular , Mutação , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Immunity ; 38(3): 437-49, 2013 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-23499489

RESUMO

RIG-I and MDA5 have emerged as key cytosolic sensors for the detection of RNA viruses and lead to antiviral interferon (IFN) production. Recent studies have highlighted the importance of posttranslational modifications for controlling RIG-I antiviral activity. However, the regulation of MDA5 signal-transducing ability remains unclear. Here, we show that MDA5 signaling activity is regulated by a dynamic balance between phosphorylation and dephosphorylation of its caspase recruitment domains (CARDs). Employing a phosphatome RNAi screen, we identified PP1α and PP1γ as the primary phosphatases that are responsible for MDA5 and RIG-I dephosphorylation and that lead to their activation. Silencing of PP1α and PP1γ enhanced RIG-I and MDA5 CARD phosphorylation and reduced antiviral IFN-ß production. PP1α- and PP1γ-depleted cells were impaired in their ability to induce IFN-stimulated gene expression, which resulted in enhanced RNA virus replication. This work identifies PP1α and PP1γ as regulators of antiviral innate immune responses to various RNA viruses, including influenza virus, paramyxovirus, dengue virus, and picornavirus.


Assuntos
RNA Helicases DEAD-box/imunologia , Imunidade Inata/imunologia , Proteína Fosfatase 1/imunologia , RNA Viral/imunologia , Animais , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/genética , Immunoblotting , Helicase IFIH1 Induzida por Interferon , Interferon beta/imunologia , Interferon beta/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Interferência de RNA , RNA Viral/genética , RNA Viral/metabolismo , Receptores Imunológicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Vero
14.
Biosci Biotechnol Biochem ; 76(3): 486-94, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22451389

RESUMO

The optimal cellular responses to DNA damage are modulated by kinase and phosphatase. The ataxia telangiectasia mutated (ATM) is a Ser/Thr kinase which is the core of the DNA damage signaling apparatus. The Ser/Thr protein phosphatase type 1 (PP1) inhibitor, tautomycetin (TC) and an antibody to the phospho-(S/T)Q sites of the ATM substrate were used to identify the common substrates for PP1 and ATM in regulating the pathway for DNA damage response. Ribosomal protein S6 (RPS6) was first identified as a substrate for PP1 and ATM. The phosphorylation at Ser247 of RPS6 was then significantly decreased by PP1-mediated dephosphorylation immediately after UV irradiation. These results suggest that PP1 specifically dephosphorylated RPS6 at phospho-Ser247 in vivo. In response to DNA damage, ATM activity was finally required for the phosphorylation of RPS6 at Ser247. We propose from these results a novel mechanism for modulating the RPS6 function by PP1 and ATM which regulates cell growth and survival in response to DNA-damage stimuli.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína S6 Ribossômica/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Especificidade de Anticorpos , Proteínas Mutadas de Ataxia Telangiectasia , Dano ao DNA , Furanos/farmacologia , Células HEK293 , Células HeLa , Humanos , Lipídeos/farmacologia , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Conformação Proteica , Proteína Fosfatase 1/química , Proteína Fosfatase 1/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta
15.
J Histochem Cytochem ; 59(8): 741-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21804078

RESUMO

Phostensin binds to the pointed ends of actin filaments and modulates actin dynamics. The genomic location of phostensin is between the HLA-C and HLA-E gene clusters on human chromosome 6, and the mRNA of this protein is predominantly distributed in the spleen, thymus, and peripheral leukocytes. However, the distribution of phostensin in leukocyte cell populations and the subcellular localization have not yet been determined. In this study, an anti-phostensin monoclonal antibody (PT2) that recognizes residues 89-124 of phostensin was prepared and used to examine the subcellular localization and distribution of phostensin in white blood cell populations and in lymphatic tissues. It was found that phostensin is mainly concentrated at the cell periphery and co-localizes with actin filaments. In addition, phostensin was abundant in helper T-lymphocytes, cytotoxic T-lymphocytes, mature monocytes, macrophages, B-lymphocytes, natural killer cells, and granulocytes as well as in the lymphatic tissues, such as the thymus, lymph nodes, and spleen. Phostensin is expressed in the mature lymphocytes of the thymic medulla but not in the immature lymphocytes of the thymic cortex. Taken together, phostensin is a ubiquitous protein in leukocytes, and it may play an important role in modulating the cellular functions of leukocytes.


Assuntos
Leucócitos Mononucleares/metabolismo , Leucócitos/metabolismo , Tecido Linfoide/metabolismo , Proteína Fosfatase 1/metabolismo , Anticorpos Monoclonais , Linhagem Celular Tumoral , Células HeLa , Humanos , Imuno-Histoquímica , Imunoprecipitação , Leucemia , Proteína Fosfatase 1/imunologia
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