RESUMO
The proinflammatory cytokines interleukin 12 (IL-12) and IL-23 connect innate responses and adaptive immune responses and are also involved in autoimmune and inflammatory diseases. Here we describe an epigenetic mechanism for regulation of the genes encoding IL-12 (Il12a and Il12b; collectively called 'Il12' here) and IL-23 (Il23a and Il12b; collectively called 'Il23' here) involving the deubiquitinase Trabid. Deletion of Zranb1 (which encodes Trabid) in dendritic cells inhibited induction of the expression of Il12 and Il23 by Toll-like receptors (TLRs), which impaired the differentiation of inflammatory T cells and protected mice from autoimmune inflammation. Trabid facilitated TLR-induced histone modifications at the promoters of Il12 and Il23, which involved deubiqutination and stabilization of the histone demethylase Jmjd2d. Our findings highlight an epigenetic mechanism for the regulation of Il12 and Il23 and establish Trabid as an innate immunological regulator of inflammatory T cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/genética , Epigênese Genética , Interleucina-12/genética , Interleucina-23/genética , Proteases Específicas de Ubiquitina/genética , Animais , Diferenciação Celular , Imunoprecipitação da Cromatina , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Immunoblotting , Imunoprecipitação , Interleucina-12/imunologia , Interleucina-23/imunologia , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Receptores Toll-Like/metabolismo , Proteases Específicas de Ubiquitina/imunologia , Dedos de Zinco/genética , Dedos de Zinco/imunologiaRESUMO
Deubiquitinases (DUBs) are a new class of drug targets, although the physiological function of only few DUBs has been characterized. Here we identified the DUB USP15 as a crucial negative regulator of T cell activation. USP15 stabilized the E3 ubiquitin ligase MDM2, which in turn negatively regulated T cell activation by targeting the degradation of the transcription factor NFATc2. USP15 deficiency promoted T cell activation in vitro and enhanced T cell responses to bacterial infection and tumor challenge in vivo. USP15 also stabilized MDM2 in cancer cells and regulated p53 function and cancer-cell survival. Our results suggest that inhibition of USP15 may both induce tumor cell apoptosis and boost antitumor T cell responses.
Assuntos
Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Células Th1/imunologia , Proteases Específicas de Ubiquitina/imunologia , Transferência Adotiva , Animais , Apoptose/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células HCT116 , Humanos , Leupeptinas/farmacologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-mdm2/genética , Evasão Tumoral , Proteína Supressora de Tumor p53/imunologia , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/imunologiaRESUMO
TBK1 is a component of the type I interferon (IFN) signaling pathway, yet the mechanisms controlling its activity and degradation remain poorly understood. Here we report that USP38 negatively regulates type I IFN signaling by targeting the active form of TBK1 for degradation in vitro and in vivo. USP38 specifically cleaves K33-linked poly-ubiquitin chains from TBK1 at Lys670, and it allows for subsequent K48-linked ubiquitination at the same position mediated by DTX4 and TRIP. Knockdown or knockout of USP38 increases K33-linked ubiquitination, but it abrogates K48-linked ubiquitination and degradation of TBK1, thus enhancing type I IFN signaling. Our findings identify an essential role for USP38 in negatively regulating type I IFN signaling, and they provide insights into the mechanisms by which USP38 regulates TBK1 ubiquitination through the NLRP4 signalosome.
Assuntos
Imunidade Inata , Interferon Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Transdução de Sinais/imunologia , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/imunologia , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Knockout , Fosforilação , Poliubiquitina/genética , Poliubiquitina/imunologia , Poliubiquitina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas/genética , Proteínas/imunologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/imunologia , Ubiquitinação , Vesiculovirus/crescimento & desenvolvimento , Vesiculovirus/imunologiaRESUMO
RIG-I plays important roles in pathogen sensing and activation of antiviral innate immune responses in response to RNA viruses. RIG-I-mediated signaling must be precisely controlled to maintain innate immune signaling homeostasis. Previous studies demonstrated that lysine 63 (K63)-linked polyubiquitination of RIG-I is vital for its activation, but the mechanisms through which RIG-I is deubiquitinated to control innate immune responses are not well understood. Here we identified USP27X as a negative regulator of antiviral signaling in response to RNA viruses through siRNA library screening. Further functional studies indicated that USP27X negatively modulated RIG-I-mediated antiviral signaling in a deubiquitinase-dependent manner. Mechanistically, we found that USP27X removed K63-linked polyubiquitin chains from RIG-I to negatively modulate type I interferon signaling. Collectively, these studies uncover a novel negative regulatory role of USP27X in targeting RIG-I to balance innate immune responses.
Assuntos
Proteína DEAD-box 58 , Imunidade Inata/genética , Transdução de Sinais , Proteases Específicas de Ubiquitina , Vírus/imunologia , Animais , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Células HeLa , Células Hep G2 , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Camundongos , Células RAW 264.7 , Receptores Imunológicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/imunologia , Ubiquitinação/genética , Ubiquitinação/imunologiaRESUMO
The ubiquitin-specific proteases (USPs) have attracted particular attention due to their multiple functions in different biological processes. USP12, a member of the USP family, has been demonstrated to exert critical roles in diverse cellular processes, including cell death, cancer and antiviral immunity. Here, we cloned a USP12 homolog from orange spotted grouper (Epinephelus coioides, E. coioides), and its roles in fish RNA virus replication were investigated. EcUSP12 contained a 1119-bp open reading frame (ORF) encoding a 372-amino acid polypeptide, which shared 100.00% and 91.32% identity with USP12 homolog of Etheostoma cragini and Homo sapiens, respectively. Sequence analysis indicated that EcUSP12 contained a conserved peptidase-C19G domain (aa 40-369). qPCR analysis showed that EcUSP12 transcript was most abundant in head kidney and spleen of grouper E. coioides. The expression of EcUSP12 was significantly upregulated in grouper spleen (GS) cells in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization analysis showed that EcUSP12 was evenly distributed throughout the cytoplasm, and mainly co-localized with endoplasmic reticulum (ER). Interestingly, during RGNNV infection, the endogenous distribution of EcUSP12 was obviously altered, and mostly overlapped with viral coat protein (CP). Co-Immunoprecipitation (Co-IP) assay indicated that EcUSP12 interacted with viral CP. In addition, overexpression of EcUSP12 significantly inhibited the replication of RGNNV in vitro, as evidenced by the decrease in viral gene transcription and protein synthesis during infection. Consistently, knockdown of EcUSP12 by small interfering RNA (siRNA) promoted the replication of RGNNV. Furthermore, EcUSP12 overexpression also increased the transcription level of inflammatory factors and interferon-related genes, including tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, interferon regulatory factor 3 (IRF3), and IRF7. Taken together, our results demonstrated that EcUSP12, as a positive regulator of IFN signaling, interacted with viral CP to inhibit virus infection.
Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Proteínas de Peixes/imunologia , Imunidade Inata , Proteases Específicas de Ubiquitina/imunologia , Sequência de Aminoácidos , Animais , Bass/imunologia , Bass/virologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Nodaviridae , Filogenia , Alinhamento de SequênciaRESUMO
During RNA virus infection, the adaptor protein MAVS recruits TRAF3 and TRAF6 to form a signalosome, which is critical to induce the production of type I interferons (IFNs) and proinflammatory cytokines. While activation of the MAVS/TRAF3/TRAF6 signalosome is well studied, the negative regulation of the signalosome remains largely unknown. Here we report that RNA viruses specifically promote the deubiquitinase OTUD1 expression by NF-κB-dependent mechanisms at the early stage of viral infection. Furthermore, OTUD1 upregulates protein levels of intracellular Smurf1 by removing Smurf1 ubiquitination. Importantly, RNA virus infection promotes the binding of Smurf1 to MAVS, TRAF3 and TRAF6, which leads to ubiquitination-dependent degradation of every component of the MAVS/TRAF3/TRAF6 signalosome and subsequent potent inhibition of IFNs production. Consistently, OTUD1-deficient mice produce more antiviral cytokines and are more resistant to RNA virus infection. Our findings reveal a novel immune evasion mechanism exploited by RNA viruses, and elucidate a negative feedback loop of MAVS/TRAF3/TRAF6 signaling mediated by the OTUD1-Smurf1 axis during RNA virus infection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imunidade Inata/imunologia , Vírus de RNA/fisiologia , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Camundongos , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/prevenção & controle , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/imunologiaRESUMO
Deubiquitinases are widely involved in the regulation of the virus-triggered type I interferon (IFN) signaling. Here, we found sea perch (Lateolabrax japonicus) ubiquitin-specific protease 5 (LjUSP5) was a negative regulatory factor of the red-spotted grouper nervous necrosis virus (RGNNV)-triggered IFN response. LjUSP5 encoded a polypeptide of 830 amino acids, containing a zinc finger UBP domain (residues 197-270 aa), two ubiquitin-associated domains (residues 593-607 aa; 628-665 aa), and one UBP domain (residues 782-807 aa), and shared the closest genetic relationship with the USP5 of Larimichthys crocea. Quantitative RT-PCR analysis showed that LjUSP5 was ubiquitously expressed and up-regulated significantly in all inspected tissues post RGNNV infection, and its transcripts significantly increased in brain, liver and kidney tissues post RGNNV infection. LjUSP5 was up-regulated in cultured LJB cells after poly I:C and RGNNV treatments. In addition, overexpression of LjUSP5 significantly inhibited the activation of zebrafish IFN 1 promoter and promoted RGNNV replication in vitro. Furthermore, LjUSP5 inhibited the activation of zebrafish IFN 1 promoter induced by key genes of retinoic acid-inducible gene I-like receptors signaling pathway. Our findings provides useful information for further elucidating the mechanism underlying NNV infection.
Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Nodaviridae/fisiologia , Filogenia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Proteases Específicas de Ubiquitina/químicaRESUMO
Adipose-derived stem cells (ASCs) are highly attractive for cell-based therapies in tissue repair and regeneration because they have multilineage differentiation capacity and are immunosuppressive. However, the detailed epigenetic mechanisms of their immunoregulatory capacity are not fully defined. In this study, we found that Mysm1 was induced in ASCs treated with inflammatory cytokines. Adipose-derived stem cells with Mysm1 knockdown exhibited attenuated immunosuppressive capacity, evidenced by less inhibition of T cell proliferation, more pro-inflammatory factor secretion and less nitric oxide (NO) production in vitro. Mysm1-deficient ASCs exacerbated inflammatory bowel diseases but inhibited tumour growth in vivo. Mysm1-deficient ASCs also showed depressed miR-150 expression. When transduced with Mysm1 overexpression lentivirus, ASCs exhibited enhanced miR-150 expression. Furthermore, Mysm1-deficient cells transduced with lentivirus containing miR-150 mimics produced less pro-inflammatory factors and more NO. Our study reveals a new role of Mysm1 in regulating the immunomodulatory activities of ASCs by targeting miR-150. These novel insights into the mechanisms through which ASCs regulate immune reactions may lead to better clinical utility of these cells.
Assuntos
Tecido Adiposo/citologia , Epigênese Genética/imunologia , MicroRNAs/imunologia , Células-Tronco/imunologia , Transativadores/imunologia , Proteases Específicas de Ubiquitina/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Interferon gama/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Óxido Nítrico/imunologia , Óxido Nítrico/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transativadores/genética , Transativadores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Retinoic acid-inducible gene I (RIG-I) is a key pattern recognition receptor that senses viral RNA and interacts with the mitochondrial adaptor MAVS, triggering a signaling cascade that results in the production of type I interferons (IFNs). This signaling axis is initiated by K63-linked ubiquitination of RIG-I mediated by the E3 ubiquitin ligase TRIM25, which promotes the interaction of RIG-I with MAVS. USP15 was recently identified as an upstream regulator of TRIM25, stabilizing the enzyme through removal of degradative K48-linked polyubiquitin, ultimately promoting RIG-I-dependent cytokine responses. Here, we show that the E6 oncoprotein of human papillomavirus type 16 (HPV16) as well as of other HPV types form a complex with TRIM25 and USP15 in human cells. In the presence of E6, the K48-linked ubiquitination of TRIM25 was markedly increased, and in line with this, TRIM25 degradation was enhanced. Our results further showed that E6 inhibited the TRIM25-mediated K63-linked ubiquitination of RIG-I and its CARD-dependent interaction with MAVS. HPV16 E6, but not E7, suppressed the RIG-I-mediated induction of IFN-ß, chemokines, and IFN-stimulated genes (ISGs). Finally, CRISPR-Cas9 gene targeting in human keratinocytes showed that the TRIM25-RIG-I-MAVS triad is important for eliciting an antiviral immune response to HPV16 infection. Our study thus identifies a novel immune escape mechanism that is conserved among different HPV strains and further indicates that the RIG-I signaling pathway plays an important role in the innate immune response to HPV infection.IMPORTANCE Persistent infection and tumorigenesis by HPVs are known to require viral manipulation of a variety of cellular processes, including those involved in innate immune responses. Here, we show that the HPV E6 oncoprotein antagonizes the activation of the cytoplasmic innate immune sensor RIG-I by targeting its upstream regulatory enzymes TRIM25 and USP15. We further show that the RIG-I signaling cascade is important for an antiviral innate immune response to HPV16 infection, providing evidence that RIG-I, whose role in sensing RNA virus infections has been well characterized, also plays a crucial role in the antiviral host response to small DNA viruses of the Papillomaviridae family.
Assuntos
Proteína DEAD-box 58/imunologia , Papillomavirus Humano 6/imunologia , Imunidade Inata , Queratinócitos/imunologia , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Transdução de Sinais/imunologia , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteases Específicas de Ubiquitina/imunologia , Proteína DEAD-box 58/genética , Células HEK293 , Papillomavirus Humano 6/genética , Humanos , Queratinócitos/patologia , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Receptores Imunológicos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Proteases Específicas de Ubiquitina/genéticaRESUMO
Dysregulation of innate immunity accompanied by excessive interferon production contributes to autoimmune disease. However, the mechanism by which the immune response is modulated in autoimmune disorders is largely unknown. Here we identified loss-of-function mutations of OTUD1 associated with multiple autoimmune diseases. Under inflammatory conditions, inducible OTUD1 acts as an immune checkpoint and blocks RIG-I-like receptors signaling. As a deubiquitinase, OTUD1 directly interacts with transcription factor IRF3 and removes the K63-linked poly-ubiquitin chains on IRF3 Lysine 98, which inhibits IRF3 nuclear translocation and transcriptional activity. In contrast, OTUD1 mutants impair its suppressive effects on IRF3 via attenuating the OTUD1 deubiquinase activity or its association with IRF3. Moreover, we found FOXO3 signaling is required for OTUD1 induction upon antigenic stimulation. Our data demonstrate that OTUD1 is involved in maintaining immune homeostasis and loss-of-function mutations of OTUD1 enhance the immune response and are associated with autoimmunity.
Assuntos
Artrite Reumatoide/genética , Colite Ulcerativa/genética , Doença de Hashimoto/genética , Lúpus Eritematoso Sistêmico/genética , Linfócitos/imunologia , Proteases Específicas de Ubiquitina/genética , Adulto , Sequência de Aminoácidos , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/imunologia , Regulação da Expressão Gênica , Células HEK293 , Doença de Hashimoto/imunologia , Doença de Hashimoto/patologia , Homeostase/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Linfócitos/patologia , Masculino , Mutação , Transporte Proteico , Receptores Imunológicos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteases Específicas de Ubiquitina/imunologiaRESUMO
Host pattern-recognition receptors (PRRs) recognize pathogen-associated molecular patterns generated by invading viruses and initiate a series of signaling cascades that lead to the activation of interferon-regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB) and subsequent induction of type I interferons (IFNs). Posttranslational modification of proteins by ubiquitin plays an essential role in mediating or regulating the virus-triggered PRRs-mediated signaling. Deubiquitination is the reversible process of ubiquitination and its role in regulating PRRs-mediated signaling has recently been explored. In this review, we first summarize the ubiquitination events in PRRs-mediated signaling that is triggered by viral nucleic acid and then focus on host and viral deubiquitinating enzymes-mediated regulation of virus-triggered signaling that modulates the activation of IRF3 and NF-κB and subsequent induction of type I IFNs.
Assuntos
Imunidade Celular/fisiologia , Imunidade Inata/fisiologia , Ubiquitinação/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Proteases Específicas de Ubiquitina/imunologia , Proteases Específicas de Ubiquitina/metabolismo , Vírus/imunologia , Vírus/patogenicidadeRESUMO
Ubiquitin-specific protease(USP), which belongs to cysteine protease, is an important member of the deubiquitinating enzyme family(DUB). USP plays an important role in the immune response against viral infections, in which it can regulate the production of type I interferon through various ways to initiate or weaken the antiviral immune response. USP2b, USP3, USP18, USP25, UL36USP and HAUSP play a role of antivirus; while USP4, USP13, USP15 and USP17 negatively regulate antiviral immune response. In this article we review the recent progress on roles of USP family in antiviral immune response.
Assuntos
Proteases Específicas de Ubiquitina/imunologia , Viroses/imunologia , Humanos , Interferon Tipo I/imunologiaRESUMO
BACKGROUND: This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. METHODS: We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. RESULTS: One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. CONCLUSIONS: The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.
Assuntos
Adenocarcinoma Folicular/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Papilar/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Adenocarcinoma Folicular/patologia , Biomarcadores Tumorais/imunologia , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/química , Neoplasias da Glândula Tireoide/patologia , Proteases Específicas de Ubiquitina/imunologiaRESUMO
BACKGROUND AND AIMS: The inflammatory bowel disease [IBD]-associated immune response is marked by excessive production of a variety of inflammatory cytokines, which are supposed to sustain and amplify the pathological process. OTUD5 is a deubiquitinating enzyme, which regulates cytokine production by both innate and adaptive immune cells. Here, we investigated the expression and role of OTUD5 in IBD. METHODS: OTUD5 expression was evaluated in mucosal samples of patients with Crohn's disease [CD], patients with ulcerative colitis [UC], and controls, as well as in mice with trinitrobenzene-sulphonic acid [TNBS]-induced colitis by real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. Moreover, OTUD5 was assessed in lamina propria mononuclear cells [LPMC] stimulated with inflammatory cytokines. TNF-α, IL-6, and IL-10 were evaluated in LPMCs of IBD patients and in colitic mice transfected with a specific OTUD5 antisense oligonucleotide [AS]. RESULTS: OTUD5 protein, but not RNA, expression was increased in inflamed ileal and colonic mucosal samples of patients with CD and patients with UC as compared with controls. In IBD, OTUD5-expressing cells were abundant in both epithelial and lamina propria compartments, and non-CD3+, HLA-DR+ LPMC were one of the major sources of the protein. OTUD5 expression was enhanced by IFN-γ through a p38/MAPK-dependent mechanism, and the AS-induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-α. CONCLUSIONS: Our data show that OTUD5 is overexpressed in both CD and UC and suggest the involvement of such a protein in the amplification of the aberrant cytokine response in IBD.
Assuntos
Citocinas/imunologia , Endopeptidases/imunologia , Doenças Inflamatórias Intestinais/imunologia , Proteases Específicas de Ubiquitina/imunologia , Animais , Biópsia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
AIMS: Asthma is characterized by chronic inflammation and airway hyperresponsiveness (AHR). It is controllable, but not curable. Ubiquitin-specific peptidase 4 (USP4) has been verified as a regulator of regulatory T (Treg) cells and Th17 cells in vitro. In this study, we aim to investigate whether USP4 could serve as a therapeutic target for asthma. MAIN METHODS: Age-matched USP4 wild-type and knockout mice received an intraperitoneal injection of 100 µg ovalbumin (OVA) mixed in 2 mg aluminum hydroxide in 1 × PBS on days 0, 7 and 14. On days 21 to 27, the mice were challenged with aerosolized 1% OVA in 1 × PBS for 30 min. Tissue histology, ELISA and flow cytometry were applied 24 h after the last OVA challenge. KEY FINDINGS: USP4 deficiency protected mice from OVA-induced AHR and decreased the production of several inflammatory cytokines in T cells in vivo. Compared to the lung cells isolated from WT mice, Usp4-/- lung cells decreased secretion of IL-4, IL-13 and IL-17A upon stimulation in vitro. Meanwhile, the percentage of CD4+Foxp3+ Treg cells was elevated, with more CCR6+Foxp3+ Treg cells accumulating in the lungs of OVA-challenged USP4 deficient mice than in their wild-type counterparts. Treatment with the USP4 inhibitor, Vialinin A, reduced inflammatory cell infiltration in the lungs of OVA-challenged mice in vivo. SIGNIFICANCE: We found USP4 deficiency contributes to attenuated airway inflammation and AHR in allergen-induced murine asthma, and Vialinin A treatment alleviates asthma pathogenesis and may serve as a promising therapeutic target for asthma.
Assuntos
Asma/imunologia , Linfócitos T Reguladores/imunologia , Proteases Específicas de Ubiquitina/imunologia , Animais , Líquido da Lavagem Broncoalveolar , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/citologia , Proteases Específicas de Ubiquitina/genéticaRESUMO
Cyclic GMP-AMP synthase (cGAS) is an essential sensor of cytosolic DNA and critically mediates innate immune responses and autoimmunity. Modulating the activity and stability of cGAS provides potential strategies for treating viral or autoimmune diseases. Here, we report that ubiquitin-specific protease 29 (USP29) deubiquitinates and stabilizes cGAS and promotes cellular antiviral responses and autoimmunity. Knockdown or knockout of USP29 severely impairs Herpes simplex virus 1 (HSV-1)- or cytosolic DNA-induced expression of type I interferons (IFNs) and proinflammatory cytokines. Consistently, Usp29m/m mice produce decreased type I IFNs and proinflammatory cytokines after HSV-1 infection and are hypersensitive to HSV-1 infection compared to the wild-type littermates. In addition, genetic ablation of USP29 in Trex1-/- mice eliminated the detectable pathological and molecular autoimmune phenotypes. Mechanistically, USP29 constitutively interacts with cGAS, deconjugates K48-linked polyubiquitin chains from cGAS and stabilizes cGAS in uninfected cells or after HSV-1 infection. Reconstitution of cGAS into Usp29-/- cells fully rescues type I IFN induction and cellular antiviral responses after HSV-1 infection. Our findings thus reveal a critical role of USP29 in the innate antiviral responses against DNA viruses and autoimmune diseases and provide insight into the regulation of cGAS.
Assuntos
Antivirais/imunologia , Doenças Autoimunes/imunologia , Herpes Simples/imunologia , Nucleotidiltransferases/imunologia , Proteases Específicas de Ubiquitina/imunologia , Animais , Células da Medula Óssea , Células HEK293 , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The immune system is not only required for preventing threats exerted by pathogens but also essential for developing immune tolerance to avoid tissue damage. This study identifies a distinct mechanism by which MYSM1 suppresses innate immunity and autoimmunity. The expression of MYSM1 is induced upon DNA virus infection and by intracellular DNA stimulation. MYSM1 subsequently interacts with STING and cleaves STING K63-linked ubiquitination to suppress cGAS-STING signaling. Notably, Mysm1-deficient mice exhibit a hyper-inflammatory response, acute tissue damage, and high mortality upon virus infection. Moreover, in the PBMCs of patients with systemic lupus erythematosus (SLE), MYSM1 production decreases, while type I interferons and pro-inflammatory cytokine expressions increase. Importantly, MYSM1 treatment represses the production of IFNs and pro-inflammatory cytokines in the PBMCs of SLE patients. Thus, MYSM1 is a critical repressor of innate immunity and autoimmunity and is thus a potential therapeutic agent for infectious, inflammatory, and autoimmune diseases.
Assuntos
Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Adulto , Animais , Doenças Autoimunes , Autoimunidade/imunologia , China , Feminino , Humanos , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Interferon Tipo I/fisiologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Nucleotidiltransferases/fisiologia , Transdução de Sinais/genética , Transativadores/genética , Transativadores/imunologia , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/imunologiaRESUMO
Th2 immune response is critical for allergic asthma pathogenesis. Molecular mechanisms for regulating Th2 immunity are still not well understood. Here we report that the ubiquitin-specific protease USP38 is crucial for Th2-mediated allergic asthma. TCR stimulation up-regulated the USP38 level, and USP38 in turn mediated the protein stabilization of JunB, a transcription factor specific for Th2 development. Consequently, USP38 was specifically required for TCR-induced production of Th2 cytokines and Th2 development both in vitro and in vivo, and USP38-deficient mice were resistant to asthma pathogenesis induced by OVA or HDM. Mechanistically, USP38 directly associated with JunB, deubiquitinated Lys-48-linked poly-ubiquitination of JunB, and consequently blocked TCR-induced JunB turnover. USP38 represents the first identified deubiquitinase specifically for Th2 immunity and the associated asthma.
Assuntos
Asma/imunologia , Células Th2/imunologia , Fatores de Transcrição/imunologia , Proteases Específicas de Ubiquitina/imunologia , Animais , Asma/genética , Asma/patologia , Citocinas/genética , Citocinas/imunologia , Camundongos , Camundongos Knockout , Poliubiquitina/genética , Poliubiquitina/imunologia , Estabilidade Proteica , Células Th2/patologia , Fatores de Transcrição/genética , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/imunologiaRESUMO
Ovarian tumor domain-containing protein 1 (OTUD1), an OTU-family deubiquitinating enzyme, has been reported to be involved in cancer progression through the regulation of p53 and SMAD7. However, the precise pathophysiological functions of OTUD1 remain elusive. Here, we report the establishment of OTUD1-specific monoclonal antibodies (mAbs), using the rat medial iliac lymph node method. The generated antibodies recognize the N-terminal portion (aa. 1-290) of human and mouse OTUD1 proteins. In addition, immunofluorescent staining and subcellular fractionation analyses using these antibodies indicated that OTUD1 is predominantly localized in the cytosol. Thus, these mAbs can be further used to elucidate cellular functions of OTUD1 and its involvement in processes such as cancer progression.
Assuntos
Anticorpos Monoclonais/imunologia , Neoplasias/imunologia , Proteases Específicas de Ubiquitina/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Humanos , Linfonodos/imunologia , Camundongos , Neoplasias/genética , Neoplasias/patologia , Ratos , Proteína Smad7/imunologia , Proteína Supressora de Tumor p53/imunologia , Proteases Específicas de Ubiquitina/antagonistas & inibidoresRESUMO
Ubiquitin-specific protease 18 (USP18) is known as an inhibition factor and has been associated with the innate immune response to pathogens. USP18 is the only deconjugating protease with specificity for interferon-stimulated gene 15 (ISG15), which is supposed to be missing in birds. To analyze the efficacy of goose USP18 (goUSP18) against Tembusu virus (TMUV) infection, we first cloned USP18 homologous cDNA from TMUV infected geese. The coding sequence was 1131 bp, and the deduced amino acid sequence shared conserved motifs with its homologues. Tissue-specific expression has shown that goUSP18 transcripts are strongly expressed in the spleen and liver of adult geese, as well as in the pancreas of goslings. Moreover, the goUSP18 transcripts were induced by goose interferons (goIFN) in goose embryo fibroblasts (GEF) and by TLR ligands in peripheral blood mononuclear cells (PBMC). Notably, goUSP18 transcripts were highly up-regulated by TMUV infection compared to the basal level in uninfected birds. Taken together, these results suggested that goUSP18 was involved in host innate immunity against TMUV infection.