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1.
Nat Commun ; 10(1): 4779, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31636267

RESUMO

Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation.


Assuntos
Linfo-Histiocitose Hemofagocítica/genética , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/genética , Adolescente , Animais , Códon sem Sentido , Consanguinidade , Ciclosporina/uso terapêutico , Eosinofilia/genética , Eosinofilia/imunologia , Homozigoto , Humanos , Imunofenotipagem , Imunossupressores/uso terapêutico , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Linfo-Histiocitose Hemofagocítica/tratamento farmacológico , Linfo-Histiocitose Hemofagocítica/imunologia , Masculino , Camundongos , Monócitos/imunologia , Receptores OX40/genética , Receptores OX40/imunologia , Receptores OX40/metabolismo , Recidiva , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Ubiquitina-Proteína Ligases/imunologia
2.
Mol Immunol ; 114: 513-523, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518855

RESUMO

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitroand interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility.


Assuntos
Epitopos/imunologia , Peptídeos/imunologia , Anticorpos de Domínio Único/imunologia , Enzimas de Conjugação de Ubiquitina/imunologia , Anticorpos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Degradação Associada com o Retículo Endoplasmático/imunologia , Células HeLa , Humanos , Células K562 , Fosforilação/imunologia , Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/imunologia
3.
Nat Commun ; 10(1): 3252, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324801

RESUMO

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play a critical role in defence against pathogens in plants and animals. However, we know very little about NLR-interacting proteins and the mechanisms that regulate NLR levels. Here, we used proximity labeling (PL) to identify the proteome proximal to N, which is an NLR that confers resistance to Tobacco mosaic virus (TMV). Evaluation of different PL methods indicated that TurboID-based PL provides more efficient levels of biotinylation than BioID and BioID2 in plants. TurboID-based PL of N followed by quantitative proteomic analysis and genetic screening revealed multiple regulators of N-mediated immunity. Interestingly, a putative E3 ubiquitin ligase, UBR7, directly interacts with the TIR domain of N. UBR7 downregulation leads to an increased amount of N protein and enhanced TMV resistance. TMV-p50 effector disrupts the N-UBR7 interaction and relieves negative regulation of N. These findings demonstrate the utility of TurboID-based PL in plants and the N-interacting proteins we identified enhance our understanding of the mechanisms underlying NLR regulation.


Assuntos
Proteínas NLR/imunologia , Proteínas de Plantas/imunologia , Receptores Imunológicos/imunologia , Coloração e Rotulagem/métodos , Tabaco/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas NLR/metabolismo , Imunidade Vegetal/imunologia , Proteínas de Plantas/metabolismo , Ligação Proteica , Proteoma/imunologia , Proteoma/metabolismo , Receptores Imunológicos/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/imunologia , Tabaco/metabolismo , Tabaco/virologia , Vírus do Mosaico do Tabaco/imunologia , Vírus do Mosaico do Tabaco/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
4.
Nature ; 571(7766): 565-569, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31316206

RESUMO

Parkinson's disease is a neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the substantia nigra compacta. Although the mechanisms that trigger the loss of dopaminergic neurons are unclear, mitochondrial dysfunction and inflammation are thought to have key roles1,2. An early-onset form of Parkinson's disease is associated with mutations in the PINK1 kinase and PRKN ubiquitin ligase genes3. PINK1 and Parkin (encoded by PRKN) are involved in the clearance of damaged mitochondria in cultured cells4, but recent evidence obtained using knockout and knockin mouse models have led to contradictory results regarding the contributions of PINK1 and Parkin to mitophagy in vivo5-8. It has previously been shown that PINK1 and Parkin have a key role in adaptive immunity by repressing presentation of mitochondrial antigens9, which suggests that autoimmune mechanisms participate in the aetiology of Parkinson's disease. Here we show that intestinal infection with Gram-negative bacteria in Pink1-/- mice engages mitochondrial antigen presentation and autoimmune mechanisms that elicit the establishment of cytotoxic mitochondria-specific CD8+ T cells in the periphery and in the brain. Notably, these mice show a sharp decrease in the density of dopaminergic axonal varicosities in the striatum and are affected by motor impairment that is reversed after treatment with L-DOPA. These data support the idea that PINK1 is a repressor of the immune system, and provide a pathophysiological model in which intestinal infection acts as a triggering event in Parkinson's disease, which highlights the relevance of the gut-brain axis in the disease10.


Assuntos
Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/fisiopatologia , Intestinos/microbiologia , Doença de Parkinson/genética , Doença de Parkinson/microbiologia , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Animais , Apresentação do Antígeno/imunologia , Autoantígenos/imunologia , Axônios/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Modelos Animais de Doenças , Neurônios Dopaminérgicos/imunologia , Neurônios Dopaminérgicos/patologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/patologia , Feminino , Intestinos/imunologia , Intestinos/patologia , Levodopa/uso terapêutico , Masculino , Camundongos , Mitocôndrias/imunologia , Mitocôndrias/patologia , Neostriado/imunologia , Neostriado/microbiologia , Neostriado/patologia , Neostriado/fisiopatologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/fisiopatologia , Proteínas Quinases/imunologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia
5.
Mol Plant Microbe Interact ; 32(11): 1487-1495, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31241412

RESUMO

Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein-protein interactions between the causal agent of apple proliferation 'Candidatus Phytoplasma mali' and its host plant. Here, we report that the 'Ca. P. mali' strain PM19 genome encodes a protein PM19_00185 that interacts with at least six different ubiquitin-conjugating enzymes (UBC; E2) of Arabidopsis thaliana. An in vitro ubiquitination assay showed that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09 and Malus domestica E2 UBC10. We show that a nonhost bacteria (Pseudomonas syringae pv. tabaci) can grow in transgenic A. thaliana plant lines expressing PM19_00185. A connection of phytoplasma effector proteins with the proteasome proteolytic pathway has been reported before. However, this is, to our knowledge, the first time that a phytoplasma effector protein with E3 ligase activity has been reported.


Assuntos
Phytoplasma , Doenças das Plantas , Ubiquitina-Proteína Ligases , Arabidopsis/enzimologia , Arabidopsis/parasitologia , Malus/parasitologia , Phytoplasma/enzimologia , Phytoplasma/genética , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
6.
Diagn Pathol ; 14(1): 47, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31109352

RESUMO

BACKGROUND: Small cell lung cancer (SCLC) is usually diagnosed in the advanced stage. It has a very poor prognosis, with no advancements in therapy in the last few decades. A recent phase 1 clinical study, using an antibody-drug conjugate directed against DLL3, showed promising results. A prerequisite for this therapy is an immunohistochemical test for DLL3 expression. The antibody used in the clinical trial was bound to a specific platform, which is not available in all pathology laboratories. In this study, the expression of DLL3 was analyzed using different DLL3 antibodies in high-grade neuroendocrine tumors of the lung and cell cultures. Additionally, correlation of DLL3 expression with Rb1 loss and TP53 mutation was evaluated. METHODS: The study cohort consisted of surgically resected cases, 24 SCLC and 29 large cell neuroendocrine carcinoma (LCNEC), from which tissue microarrays (TMAs) were constructed. The validation cohort included 46 SCLC samples, mostly small biopsies. Additionally, well-characterized SCLC cell lines were used. Immunohistochemical analysis was performed using four different DLL3 antibodies, as well as TP53 and Rb1 antibodies. Expression was evaluated microscopically and manually scored. RESULTS: The comparison of all DLL3 antibodies showed poor results for the overall agreement, as well as positive and negative agreement. Differences were observed regardless of the applied cut-off values and the tumor type. The antibody used in the clinical trial was the only which always positively stained the tumor cells obtained from cell cultures with known DLL3 expression and was negative on cells that did not express DLL3. There was no correlation between p53 and DLL3 expression in SCLC and LCNEC. RB1 loss in SCLC showed statistical significant correlation with the DLL3 positivity (p = 0.037), while no correlation was found in LCNEC. CONCLUSION: The DLL3 antibody used in the clinical trial demonstrated superiority in the detection of DLL3 expression. Cell cultures, which can be used for DLL3 antibodies as positive and negative probes, were established. Evidence of DLL3 expression in high proportions of patients with LCNEC might provide basis for studies of new therapy options in this group of patients.


Assuntos
Anticorpos/imunologia , Carcinoma de Células Grandes/diagnóstico , Carcinoma Neuroendócrino/diagnóstico , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neoplasias Pulmonares/diagnóstico , Proteínas de Membrana/imunologia , Tumores Neuroendócrinos/diagnóstico , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Células Grandes/patologia , Carcinoma Neuroendócrino/patologia , Linhagem Celular Tumoral , Estudos de Coortes , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Mutação , Tumores Neuroendócrinos/patologia , Prognóstico , Proteínas de Ligação a Retinoblastoma/imunologia , Proteínas de Ligação a Retinoblastoma/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
7.
Nat Commun ; 10(1): 1492, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940817

RESUMO

Accumulating evidence points to an important role for the gut microbiome in anti-tumor immunity. Here, we show that altered intestinal microbiota contributes to anti-tumor immunity, limiting tumor expansion. Mice lacking the ubiquitin ligase RNF5 exhibit attenuated activation of the unfolded protein response (UPR) components, which coincides with increased expression of inflammasome components, recruitment and activation of dendritic cells and reduced expression of antimicrobial peptides in intestinal epithelial cells. Reduced UPR expression is also seen in murine and human melanoma tumor specimens that responded to immune checkpoint therapy. Co-housing of Rnf5-/- and WT mice abolishes the anti-tumor immunity and tumor inhibition phenotype, whereas transfer of 11 bacterial strains, including B. rodentium, enriched in Rnf5-/- mice, establishes anti-tumor immunity and restricts melanoma growth in germ-free WT mice. Altered UPR signaling, exemplified in Rnf5-/- mice, coincides with altered gut microbiota composition and anti-tumor immunity to control melanoma growth.


Assuntos
Proliferação de Células , Microbioma Gastrointestinal , Melanoma/imunologia , Melanoma/microbiologia , Proteínas de Membrana/deficiência , Ubiquitina-Proteína Ligases/deficiência , Animais , Peptídeos Catiônicos Antimicrobianos/imunologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Humanos , Intestinos/imunologia , Intestinos/microbiologia , Melanoma/enzimologia , Melanoma/fisiopatologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Resposta a Proteínas não Dobradas
8.
Biochem Biophys Res Commun ; 509(3): 700-706, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30611571

RESUMO

Nuclear factor-κB (NF-κB) is a crucial transcription factor family involved in the regulation of immune and inflammatory responses and cell survival. The linear ubiquitin chain assembly complex (LUBAC), composed of the HOIL-1L, HOIP, and SHARPIN subunits, specifically generates Met1-linked linear ubiquitin chains through the ubiquitin ligase activity in HOIP, and activates the NF-κB pathway. We recently identified a chemical inhibitor of LUBAC, which we named HOIPIN-1 (HOIP inhibitor-1). To improve the potency of HOIPIN-1, we synthesized 7 derivatives (HOIPIN-2∼8), and analyzed their effects on LUBAC and NF-κB activation. Among them, HOIPIN-8 suppressed the linear ubiquitination activity by recombinant LUBAC at an IC50 value of 11 nM, corresponding to a 255-fold increase over that of HOIPIN-1. Furthermore, as compared with HOIPIN-1, HOIPIN-8 showed 10-fold and 4-fold enhanced inhibitory activities on LUBAC- and TNF-α-induced NF-κB activation respectively, without cytotoxicity. These results indicated that HOIPIN-8 is a powerful tool to explore the physiological functions of LUBAC.


Assuntos
Anti-Inflamatórios/farmacologia , NF-kappa B/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Células A549 , Anti-Inflamatórios/química , Citocinas/antagonistas & inibidores , Citocinas/imunologia , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , NF-kappa B/imunologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/imunologia , Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/efeitos dos fármacos
9.
BMC Cancer ; 19(1): 76, 2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651076

RESUMO

BACKGROUND: Invasive micropapillary carcinoma (IMPC) of the breast is characterized by its unique morphology and frequent nodal metastasis. However, the mechanism for development of this unique subtype has not been clearly elucidated. The aim of this study was to obtain a better understanding of IMPC. METHODS: Using representative cases of mixed IMPC, mRNA expression in the micropapillary area and usual invasive area was compared. Then, immunohistochemical analyses for 294 cases (76 invasive carcinomas with a micropapillary feature [ICMF] and 218 invasive carcinomas without a micropapillary feature [ICNMF]) were conducted. Clinicopathological analyses were also studied. RESULTS: DNA microarray analyses for mixed IMPC showed that BC-1514 (C21orf118) was commonly upregulated in the micropapillary area. CAMK2N1, CD1d, PJA2, RPL5, SAMD13, TCF4, and TXNIP were commonly downregulated in the micropapillary area. Immunohistochemically, we confirmed that BC-1514 was more upregulated in ICMF than in ICNMF. CD1d and PJA2 were more downregulated in ICMF than ICNMF. All patients with cases of PJA2 overexpression survived without cancer recurrence during the follow-up period, although the differences for disease-free (p = 0.153) or overall survival (p = 0.272) were not significant. CONCLUSIONS: The CD1d- and PJA2-related tumour microenvironment might be crucial for IMPC. Further study of the immune microenvironment and micropapillary features is warranted.


Assuntos
Antígenos CD1d/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Papilar/patologia , Ubiquitina-Proteína Ligases/metabolismo , Antígenos CD1d/imunologia , Mama/imunologia , Mama/patologia , Mama/cirurgia , Neoplasias da Mama/imunologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/imunologia , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/cirurgia , Carcinoma Papilar/imunologia , Carcinoma Papilar/mortalidade , Carcinoma Papilar/cirurgia , Regulação para Baixo , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sobrevida , Análise Serial de Tecidos , Microambiente Tumoral , Ubiquitina-Proteína Ligases/imunologia , Regulação para Cima
10.
Mol Immunol ; 105: 131-136, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30504095

RESUMO

NDR/LATS kinase family are conserved from yeast to man and their roles in inflammation remains largely unknown. In the present study, we show that knockdown of NDR2 significantly increases IL-17-induced IL-6, CXCL2 and CCL20 expression in Hela and HT-29 cells. Knockdown of NDR2 enhances IL-17-induced MAPK and NF-κB activation. NDR2 interacts with E3 ubiquitin protein ligase Smurf1, promotes Smurf1-mediated K48-linked ubiquitination of MEKK2 and inhibits expression of MEKK2. Consistently, knockdown of Smurf1 increases IL-17-induced IL-6, CXCL2 and CCL20 expression. On the other hand, overexpression of MEKK2 increases IL-17-induced IL-6 expression. These results suggest that NDR2 may play important roles in IL-17-associated inflammation by promoting Smurf1-mediated MEKK2 ubiquitination and degradation.


Assuntos
Interleucina-17/imunologia , MAP Quinase Quinase Quinases/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteólise , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/imunologia , Células HEK293 , Células HT29 , Células HeLa , Humanos , Interleucina-17/genética , MAP Quinase Quinase Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
11.
Cerebellum ; 18(2): 245-254, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30350014

RESUMO

To describe autoantibodies (Abs) against tripartite motif-containing (TRIM) protein 9 and 67 in two patients with paraneoplastic cerebellar degeneration (PCD) associated with lung adenocarcinoma. Abs were characterized using immunohistochemistry, Western blotting, cultures of murine cortical, and hippocampal neurons, immunoprecipitation, mass spectrometry, knockout mice for Trim9 and 67, and cell-based assay. Control samples included sera from 63 patients with small cell lung cancer without any paraneoplastic neurological syndrome, 36 patients with lung adenocarcinoma and PNS, CSF from 100 patients with autoimmune encephalitis, and CSF from 165 patients with neurodegenerative diseases. We found Abs targeting TRIM9 and TRIM67 at high concentration in the serum and the cerebrospinal fluid (CSF) of a 78-year-old woman and a 65-year-old man. Both developed subacute severe cerebellar ataxia. Brain magnetic resonance imaging found no abnormality and no cerebellar atrophy. Both had CSF inflammation with mild pleiocytosis and a few oligoclonal bands. We identified a pulmonary adenocarcinoma, confirming the paraneoplastic neurological syndrome in both patients. They received immunomodulatory and cancer treatments without improvement of cerebellar ataxia, even though both were in remission of their cancer (for more than 10 years in one patient). Anti-TRIM9 and anti-TRIM67 Abs were specific to these two patients. All control serum and CSF samples tested were negative for anti-TRIM9 and 67. Anti-TRIM9 and anti-TRIM67 Abs appeared to be specific biomarkers of PCD and should be added to the panel of antigens tested when this is suspected.


Assuntos
Autoanticorpos/sangue , Autoanticorpos/líquido cefalorraquidiano , Encéfalo/imunologia , Proteínas do Citoesqueleto/imunologia , Proteínas do Tecido Nervoso/imunologia , Degeneração Paraneoplásica Cerebelar/imunologia , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , Adenocarcinoma/imunologia , Idoso , Animais , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Encefalite/imunologia , Feminino , Doença de Hashimoto/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/imunologia , Degeneração Paraneoplásica Cerebelar/diagnóstico por imagem , Degeneração Paraneoplásica Cerebelar/terapia , Carcinoma de Pequenas Células do Pulmão/imunologia
12.
Mol Immunol ; 105: 76-85, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30496979

RESUMO

Activation of NLRP3 inflammasomes is crucial in the pathological process of Ulcerative colitis (UC), which could be negatively regulated by PINK1/Parkin-driven mitophagy. Palmatine is a herb derived isoquinoline alkaloid with potent anti-inflammatory and anti-bacteria activities. In present study, we evaluated the effect of palmatine on dextran sulfate sodium (DSS)-induced mice colitis and examined whether its effect is exerted by promoting mitophagy-mediated NLRP3 inflammasome inactivation. The result showed that palmatine (40, 100 mg/kg) significantly prevented bodyweight loss and colonic shortening in DSS mice, and reduced the disease activity index and histopathologic score. The levels of MPO, IL-1ß, TNF-α and the number of F4/80+ cells in colon of DSS mice were remarkably decreased by palmatine. Moreover, palmatine suppressed NLRP3 inflammasomes activation, but enhanced the expression of the mitophagy-related proteins involving LC3, PINK1 and Parkin in colonic tissue of DSS mice. These effects was consistent with the in vitro data revealing that palmatine inhibited the activation of NLRP3 inflammasomes, while promoted the expression and mitochondrial recruitment of PINK1 and Parkin in THP-1 cell differentiated macrophages. Furthermore, the effect of palmatine on THP-1 cells was neutralized by a mitophagy inhibitor Cyclosporin A (CsA) and PINK1-siRNA. In parallel, CsA significantly attenuated the therapeutic effect of palmatine in DSS mice, illustrating that the anti-colitis effect of palmatine is closely related to mitophagy. Taken together, the current results demonstrated that palmatine protected mice against DSS-induced colitis by facilitating PINK1/Parkin-driven mitophagy and thus inactivating NLRP3 inflammasomes in macrophage.


Assuntos
Colite , Sulfato de Dextrana/toxicidade , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Ácido Palmítico/farmacologia , Animais , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Colite/prevenção & controle , Ciclosporina/farmacologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos BALB C , Proteínas Quinases/imunologia , Células THP-1 , Ubiquitina-Proteína Ligases/imunologia
13.
Front Immunol ; 9: 2882, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30574150

RESUMO

Macrophages form an important component of the innate immune system and serve as first responders against invading pathogens. While pathways critical for initiation of inflammatory responses between macrophages and other LysM+ myeloid cells are largely similar, it remains unknown whether a specific pathway has differential effects on inflammatory responses mediated between these cells. Recent studies demonstrated that depletion of SAG (Sensitive to Apoptosis Gene), an E3 ubiquitin ligase, blocked inflammatory responses generated by macrophages and dendritic cells in response to LPS in cell culture settings. However, the in vivo role of Sag on modulation of macrophages and neutrophil is not known. Here we generated LysM-Cre/Sag fl/fl mice with selective Sag deletion in myeloid lineage, and found that in contrast to in vitro observations, LysM-Cre/Sag fl/fl mice showed increased serum levels of proinflammatory cytokines and enhanced mortality in response to LPS. Interestingly, while Sag -/- macrophages released less proinflammatory cytokines, Sag -/- neutrophils released more. Mechanistically, expression of a list of genes response to LPS was significantly altered in bone marrow cells from LysM-Cre +/Sag fl/fl mice after LPS challenge. Specifically, induction by LPS of myeloperoxidase (Mpo), a key neutrophil enzyme, and Elane, neutrophil expressed elastase, was significantly decreased upon Sag depletion. Collectively, our study revealed that Sag plays a differential role in the activation of macrophages and neutrophils.


Assuntos
Diferenciação Celular/imunologia , Macrófagos/imunologia , Células Progenitoras Mieloides/metabolismo , Neutrófilos/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Elastase de Leucócito/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Peroxidase/metabolismo , Cultura Primária de Células , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia
14.
Viruses ; 10(12)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30558248

RESUMO

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling.


Assuntos
Proteína DEAD-box 58/genética , Vírus Sincicial Respiratório Humano/fisiologia , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas não Estruturais Virais/genética , Células A549 , Linhagem Celular , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Reação em Cadeia da Polimerase , Ligação Proteica , Vírus Sincicial Respiratório Humano/genética , Transdução de Sinais , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/imunologia , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/metabolismo
15.
Cell Death Dis ; 9(12): 1178, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518749

RESUMO

Cutaneous T-cell lymphoma is a group of incurable extranodal non-Hodgkin lymphomas that develop from the skin-homing CD4+ T cell. Mycosis fungoides and Sézary syndrome are the most common histological subtypes. Although next-generation sequencing data provided significant advances in the comprehension of the genetic basis of this lymphoma, there is not uniform consensus on the identity and prevalence of putative driver genes for this heterogeneous group of tumors. Additional studies may increase the knowledge about the complex genetic etiology characterizing this lymphoma. We used SNP6 arrays and GISTIC algorithm to prioritize a list of focal somatic copy-number alterations in a dataset of multiple sequential samples from 21 Sézary syndrome patients. Our results confirmed a prevalence of significant focal deletions over amplifications: single well-known tumor suppressors, such as TP53, PTEN, and RB1, are targeted by these aberrations. In our cohort, ZEB1 (TCF8, ZFHX1A) spans a deletion having the highest level of significance. In a larger group of 43 patients, we found that ZEB1 is affected by deletions and somatic inactivating mutations in 46.5% of cases; also, we found potentially relevant ZEB1 germline variants. The survival analysis shows a worse clinical course for patients with ZEB1 biallelic inactivation. Multiple abnormal expression signatures were found associated with ZEB1 depletion in Sézary patients we verified that ZEB1 exerts a role in oxidative response of Sézary cells. Our data confirm the importance of deletions in the pathogenesis of cutaneous T-cell lymphoma. The characterization of ZEB1 abnormalities in Sézary syndrome fulfils the criteria of a canonical tumor suppressor gene. Although additional confirmations are needed, our findings suggest, for the first time, that ZEB1 germline variants might contribute to the risk of developing this disease. Also, we provide evidence that ZEB1 activity in Sézary cells, influencing the reactive oxygen species production, affects cell viability and apoptosis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Síndrome de Sézary/genética , Neoplasias Cutâneas/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Variações do Número de Cópias de DNA , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/imunologia , Polimorfismo de Nucleotídeo Único , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/imunologia , Síndrome de Sézary/imunologia , Síndrome de Sézary/mortalidade , Síndrome de Sézary/patologia , Pele/imunologia , Pele/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Análise de Sobrevida , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/deficiência , Homeobox 1 de Ligação a E-box em Dedo de Zinco/imunologia
16.
J Biol Chem ; 293(52): 20099-20111, 2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30389786

RESUMO

The CD4+CD25+FOXP3+ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-γ, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.


Assuntos
Fatores de Transcrição Forkhead/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Linfócitos T Reguladores/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/imunologia , Regulação para Cima/imunologia , Intervalo Livre de Doença , Células HEK293 , Humanos , Células Jurkat , Estabilidade Proteica , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Linfócitos T Reguladores/patologia
17.
Nat Commun ; 9(1): 4883, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451854

RESUMO

Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4+ T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.


Assuntos
Efeito Espectador , Linfócitos T CD4-Positivos/microbiologia , Linhagem da Célula/imunologia , Células Dendríticas/microbiologia , Evasão da Resposta Imune , Salmonella typhimurium/patogenicidade , Imunidade Adaptativa , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Monócitos/microbiologia , Cultura Primária de Células , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/imunologia , Transdução de Sinais , Análise de Célula Única , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia
18.
Stem Cell Res Ther ; 9(1): 273, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30359308

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells (iTregs) in specific inflammatory contexts. Stable Foxp3 expression plays a major role in the phenotypic and functional stability of iTregs. However, how MSCs induce stable Foxp3 expression remains unknown. METHODS: We first investigated the role of cell-cell contact and cytokine secretion by bone marrow-derived MSCs (BM-MSCs) on the induction, stability, and suppressive functions of Tregs under various experimental conditions that lead to Foxp3 generation by flow cytometry and ELISA respectively. Second, we studied the effect of MSCs on TRAF6, GRAIL, USP7, STUB1, and UBC13 mRNA expression in CD4+ T cells in correlation with the suppressive function of iTregs by real-time PCR; also, we investigated Foxp3 Treg-specific demethylated region (TSDR) methylation in correlation with Foxp3 stability by the high-resolution melting technique. Third, we studied the effect of ex-vivo-expanded BM-MSCs on the induction of transplant tolerance in a model of fully allogeneic skin transplantation. We further analyzed the cytokine secretion patterns in grafted mice as well as the mRNA expression of ubiquitination genes in CD4+ T cells collected from the spleens of protected mice. RESULTS: We found that in-vitro MSC-induced Tregs express high mRNA levels of ubiquitination genes such as TRAF6, GRAIL, and USP7 and low levels of STUB1. Moreover, they have enhanced TSDR demethylation. Infusion of MSCs in a murine model of allogeneic skin transplantation prolonged allograft survival. When CD4+ T cells were harvested from the spleens of grafted mice, we observed that mRNA expression of the Foxp3 gene was elevated. Furthermore, Foxp3 mRNA expression was associated with increased TRAF6, GRAIL, UBC13, and USP7 and decreased STUB1 mRNA levels compared with the levels observed in vitro. CONCLUSIONS: Our data suggest a possible ubiquitination mechanism by which MSCs convert Tconvs to suppressive and stable iTregs.


Assuntos
Antígenos CD4/imunologia , Fatores de Transcrição Forkhead/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Células-Tronco Mesenquimais/imunologia , Transplante de Pele , Linfócitos T Reguladores/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Biomarcadores/sangue , Antígenos CD4/genética , Técnicas de Cocultura , Desmetilação , Feminino , Fêmur/citologia , Fêmur/imunologia , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Subunidade alfa de Receptor de Interleucina-2/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia , Linfócitos T Reguladores/citologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologia , Tíbia/citologia , Tíbia/imunologia , Transplante Homólogo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/imunologia , Ubiquitinação
19.
Cell Rep ; 25(1): 29-37.e3, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30282035

RESUMO

Plant growth and development and outcomes of plant-microbe interactions are defined by coordinated responses to seasonal signals. The mechanisms that control the coordinated regulation of growth and immunity are not well understood. Here, we show that a common signaling module integrates environmental signals, such as photoperiod and temperature, to regulate the growth-defense balance. Key light-signaling components De-Etiolated 1 (DET1) and Constitutive Photomorphogenic 1 (COP1) negatively regulate immunity and are essential for immune modulation by photoperiod and temperature. Our results show that this is regulated by the transcription factor Phytochrome Interacting Factor 4 (PIF4), suggesting that the DET1/COP1-PIF4 module acts as a central hub for the control of growth and immunity in response to seasonal signals. These findings provide a regulatory framework for environmental signal integration.


Assuntos
Proteínas de Arabidopsis/imunologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fotoperíodo , Doenças das Plantas/imunologia , Transdução de Sinais/imunologia , Temperatura Ambiente , Ubiquitina-Proteína Ligases/metabolismo
20.
Front Immunol ; 9: 2047, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319601

RESUMO

The role of autoimmunity in Parkinson's disease (PD), as one of the most popular research subjects, has been intensively investigated in recent years. Although the ultimate cause of PD is unknown, one major area of interest remains identifying new therapeutic targets and options for patients suffering from PD. Herein, we present a comprehensive review of the impacts of autoimmunity in neurodegenerative diseases, especially PD, and we have composed a logical argument to substantiate that autoimmunity is actively involved in the pathogenesis of PD through several proteins, including α-synuclein, DJ-1, PINK1, and Parkin, as well as immune cells, such as dendritic cells, microglia, T cells, and B cells. Furthermore, a detailed analysis of the relevance of autoimmunity to the clinical symptoms of PD provides strong evidence for the close correlation of autoimmunity with PD. In addition, the previously identified relationships between other autoimmune diseases and PD help us to better understand the disease pattern, laying the foundation for new therapeutic solutions to PD. In summary, this review aims to integrate and present currently available data to clarify the pathogenesis of PD and discuss some controversial but innovative research perspectives on the involvement of autoimmunity in PD, as well as possible novel diagnostic methods and treatments based on autoimmunity targets.


Assuntos
Autoantígenos/genética , Autoimunidade/genética , Encéfalo/imunologia , Doença de Parkinson/imunologia , Animais , Autoantígenos/imunologia , Linfócitos B/imunologia , Encéfalo/citologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Microglia/imunologia , Mutação , Doença de Parkinson/diagnóstico , Doença de Parkinson/genética , Doença de Parkinson/terapia , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/imunologia , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , alfa-Sinucleína/genética , alfa-Sinucleína/imunologia
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