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1.
Proc Natl Acad Sci U S A ; 117(28): 16567-16578, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32606244

RESUMO

Malaria infection induces complex and diverse immune responses. To elucidate the mechanisms underlying host-parasite interaction, we performed a genetic screen during early (24 h) Plasmodium yoelii infection in mice and identified a large number of interacting host and parasite genes/loci after transspecies expression quantitative trait locus (Ts-eQTL) analysis. We next investigated a host E3 ubiquitin ligase gene (March1) that was clustered with interferon (IFN)-stimulated genes (ISGs) based on the similarity of the genome-wide pattern of logarithm of the odds (LOD) scores (GPLS). March1 inhibits MAVS/STING/TRIF-induced type I IFN (IFN-I) signaling in vitro and in vivo. However, in malaria-infected hosts, deficiency of March1 reduces IFN-I production by activating inhibitors such as SOCS1, USP18, and TRIM24 and by altering immune cell populations. March1 deficiency increases CD86+DC (dendritic cell) populations and levels of IFN-γ and interleukin 10 (IL-10) at day 4 post infection, leading to improved host survival. T cell depletion reduces IFN-γ level and reverse the protective effects of March1 deficiency, which can also be achieved by antibody neutralization of IFN-γ. This study reveals functions of MARCH1 (membrane-associated ring-CH-type finger 1) in innate immune responses and provides potential avenues for activating antimalaria immunity and enhancing vaccine efficacy.


Assuntos
Malária/imunologia , Plasmodium yoelii/fisiologia , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Parasita , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Malária/enzimologia , Malária/genética , Malária/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmodium yoelii/imunologia , Ubiquitina-Proteína Ligases/genética
2.
Molecules ; 25(12)2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32604797

RESUMO

Viruses can be spread from one person to another; therefore, they may cause disorders in many people, sometimes leading to epidemics and even pandemics. New, previously unstudied viruses and some specific mutant or recombinant variants of known viruses constantly appear. An example is a variant of coronaviruses (CoV) causing severe acute respiratory syndrome (SARS), named SARS-CoV-2. Some antiviral drugs, such as remdesivir as well as antiretroviral drugs including darunavir, lopinavir, and ritonavir are suggested to be effective in treating disorders caused by SARS-CoV-2. There are data on the utilization of antiretroviral drugs against SARS-CoV-2. Since there are many studies aimed at the identification of the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) infection and the development of novel therapeutic approaches against HIV-1, we used HIV-1 for our case study to identify possible molecular pathways shared by SARS-CoV-2 and HIV-1. We applied a text and data mining workflow and identified a list of 46 targets, which can be essential for the development of infections caused by SARS-CoV-2 and HIV-1. We show that SARS-CoV-2 and HIV-1 share some molecular pathways involved in inflammation, immune response, cell cycle regulation.


Assuntos
Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/metabolismo , Mineração de Dados/métodos , Infecções por HIV/epidemiologia , Infecções por HIV/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/metabolismo , Anti-Inflamatórios/uso terapêutico , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Bases de Dados Genéticas , Regulação da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Inflamação , Interferons/genética , Interferons/imunologia , Interleucinas/genética , Interleucinas/imunologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/imunologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia
3.
PLoS Pathog ; 16(5): e1008618, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453758

RESUMO

The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) - a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling - to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.


Assuntos
Proteínas de Ligação a DNA/imunologia , Regulação para Baixo/imunologia , Instabilidade Genômica/imunologia , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Leucemia-Linfoma de Células T do Adulto/imunologia , Proteínas de Neoplasias/imunologia , Ubiquitina-Proteína Ligases/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Reparo do DNA/imunologia , Proteínas de Ligação a DNA/genética , Produtos do Gene tax/genética , Produtos do Gene tax/imunologia , Infecções por HTLV-I/genética , Infecções por HTLV-I/patologia , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas de Neoplasias/genética , Ubiquitina-Proteína Ligases/genética
4.
PLoS Pathog ; 16(4): e1008458, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339205

RESUMO

The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by LPS or IL-1ß in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.


Assuntos
Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Proteínas Nucleares/imunologia , Fatores de Transcrição/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Bactérias Gram-Negativas/fisiologia , Células HeLa , Humanos , Imunidade Inata , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
5.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32295906

RESUMO

ND10 nuclear bodies, as part of the intrinsic defenses, impose repression on incoming DNA. Infected cell protein 0 (ICP0), an E3 ubiquitin ligase of herpes simplex virus 1 (HSV-1), can derepress viral genes by degrading ND10 organizers to disrupt ND10. These events are part of the initial tug of war between HSV-1 and host, which determines the ultimate outcome of infection. Previously, we reported that ICP0 differentially recognizes promyelocytic leukemia (PML) isoforms. ICP0 depends on a SUMO-interaction motif located at residues 362 to 364 (SIM362-364) to trigger the degradation of PML isoforms II, IV, and VI, while using a bipartite sequence flanking the RING domain to degrade PML I. In this study, we investigated how the SUMO-SIM interaction regulates the degradation of PML II and PML II-associated proteins in ND10. We found that (i) the same regulatory mechanism for PML II degradation was detected in cells permissive or nonpermissive to the ICP0-null virus; (ii) the loss of a single SIM362-364 motif was restored by the presence of four consecutive SIMs from RNF4, but was not rescued by only two of the RNF4 SIMs; (iii) the loss of three C-terminal SIMs of ICP0 was fully restored by four RNF4 SIMs and also partially rescued by two RNF4 SIMs; and (iv) a PML II mutant lacking both lysine SUMOylation and SIM was not recognized by ICP0 for degradation, but was localized to ND10 and mitigated the degradation of other ND10 components, leading to delayed viral production. Taken together, SUMO regulates ICP0 substrate recognition via multiple fine-tuned mechanisms in HSV-1 infection.IMPORTANCE HSV-1 ICP0 is a multifunctional immediate early protein key to effective replication in the HSV-1 lytic cycle and reactivation in the latent cycle. ICP0 transactivates gene expression by orchestrating an overall mitigation in host intrinsic/innate restrictions. How ICP0 coordinates its multiple active domains and its diverse protein-protein interactions is a key question in understanding the HSV-1 life cycle and pathogenesis. The present study focuses on delineating the regulatory effects of the SUMO-SIM interaction on ICP0 E3 ubiquitin ligase activity regarding PML II degradation. For the first time, we discovered the importance of multivalency in the PML II-ICP0 interaction network and report the involvement of different regulatory mechanisms in PML II recognition by ICP0 in HSV-1 infection.


Assuntos
Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteínas Imediatamente Precoces/imunologia , Proteínas Nucleares/imunologia , Proteína da Leucemia Promielocítica/imunologia , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/imunologia , Ubiquitina-Proteína Ligases/imunologia , Linhagem Celular Tumoral , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Mutação , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteólise , Transdução de Sinais , Sumoilação , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
6.
Mol Immunol ; 121: 195-203, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32298923

RESUMO

Cells recognize virus nucleic acid by pattern recognition receptors (PRRs), virus involve in the activation of signaling cascade of variable adaptor proteins, TANK-binding kinase1(TBK1)/ inhibitor of nuclear factor kappa-B kinase subunit epsilon(IKKi) complex, IκB kinase(IKKs) to trigger activation of transcription factor, interferon regulatory factor 3/7(IRF3/7), ultimately, leading to the production of type I interferon and exert anti-viral effects. In this study, E3 ubiquitin ligase ankyrin repeat and SOCS box-containing 8(ASB8) interacted with TBK1/IKKi and phosphorylation modification of ASB8 at site of Ser17 to further strengthen its ubiquitination activity were verified. Conversely, phosphorylated ASB8 accelerate K48-linked ubiquitination and degradation of TBK1/IKKi, which further reduces phosphorylation level of IRF3 and inhibits production of IFN-ß. At the same time, a new bridge molecule Leucine-rich repeat containing protein 10B(LRRC10B) upregulated after viral infection are involved in the formation and interaction with ASB8-TBK1/IKKi complex was reported. Our study reveals a new mechanism of ubiquitin ligase ASB8 modulating antiviral innate immunity by altering stability of TBK1/IKKi kinase complex.


Assuntos
Quinase I-kappa B/metabolismo , Interferon beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/imunologia , Fosforilação/imunologia , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/imunologia
7.
PLoS Pathog ; 16(3): e1008387, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126128

RESUMO

Mediator of IRF3 activation (MITA, also named as STING/ERIS/MPYS/TMEM173), is essential to DNA virus- or cytosolic DNA-triggered innate immune responses. In this study, we demonstrated the negative regulatory role of RING-finger protein (RNF) 90 in innate immune responses targeting MITA. RNF90 promoted K48-linked ubiquitination of MITA and its proteasome-dependent degradation. Overexpression of RNF90 inhibited HSV-1- or cytosolic DNA-induced immune responses whereas RNF90 knockdown had the opposite effects. Moreover, RNF90-deficient bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMMs) and mouse embryonic fibroblasts (MEFs) exhibited increased DNA virus- or cytosolic DNA-triggered signaling and RNF90 deficiency protected mice from DNA virus infection. Taken together, our findings suggested a novel function of RNF90 in innate immunity.


Assuntos
Herpesvirus Humano 1/imunologia , Imunidade Inata , Proteínas de Membrana/imunologia , Proteólise , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Herpesvirus Humano 1/genética , Macrófagos/imunologia , Macrófagos/virologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
8.
Sci Immunol ; 5(43)2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980485

RESUMO

Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans-but not laboratory mice-have a second, potent, STING-independent DNA sensing pathway (SIDSP). We identify human DNA-dependent protein kinase (DNA-PK) as the sensor of this pathway and demonstrate that DNA-PK activity drives a robust and broad antiviral response. We show that the E1A oncoprotein of human adenovirus 5 and the ICP0 protein of herpes simplex virus 1 block this response. We found heat shock protein HSPA8/HSC70 as a target for inducible phosphorylation in the DNA-PK antiviral pathway. Last, we demonstrate that DNA damage and detection of foreign DNA trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, a specific downstream target, and viral antagonists of a SIDSP in human cells.


Assuntos
Proteína Quinase Ativada por DNA/imunologia , Adenoviridae , Proteínas E1A de Adenovirus/imunologia , Animais , Linhagem Celular , Herpes Simples/imunologia , Herpesvirus Humano 1 , Humanos , Proteínas Imediatamente Precoces/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Ubiquitina-Proteína Ligases/imunologia
9.
J Immunol ; 204(5): 1322-1333, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31996460

RESUMO

With the development of liver surgery, ischemia-reperfusion (IR) injury has received increasing attention. Roquin-1 has been shown to play an important role in innate immune and immune balance. We demonstrate that Roquin-1 expression increased at 1 h after IR and then decreased in C57B/L mice. The immunofluorescence double-label showed that Roquin-1 was mainly expressed in macrophages (mø). Furthermore, we used clodronate liposomes to remove mø, and injected the bone marrow-derived mø (BMDM) through the tail vein in 1 h before IR. We found that liver IR injury was aggravated by Roquin-1 interference. The results of PCR and ELISA suggested that after interference with Roquin-1, mø increased toward M1 and decreased toward M2. Then, interference with Roquin-1 promoted the polarization of mø to M1 and inhibited the polarization of M2. By Western blot technology and AMPKα and mTOR inhibitors, we found that Roquin-1 promotes the phosphorylation of mTOR and STAT3 by inhibiting the phosphorylation of AMPKα. We used AICAR to activate AMPKα in mø and found that the level of ubiquitination of AMPKα was decreased after activation of AMPKα. Furthermore, by bioinformatics methods, we identified potential ubiquitination sites on AMPKα. By the point mutation experiments in vitro, we confirmed that the ubiquitination of these sites is regulated by Roquin-1. Meanwhile, Roquin-1 interference inhibited the activation and function of AMPKα. This topic describes the protection of liver IR injury by Roquin-1 and discusses its main mechanism for regulating AMPKα activity through ubiquitination and affecting the polarization of mø.


Assuntos
Hepatopatias/imunologia , Fígado/imunologia , Macrófagos/imunologia , Traumatismo por Reperfusão/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/imunologia , Animais , Ativação Enzimática/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Fígado/patologia , Hepatopatias/genética , Hepatopatias/patologia , Macrófagos/patologia , Masculino , Camundongos , Mutação Puntual , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Ubiquitinação/imunologia
10.
PLoS One ; 15(1): e0226928, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914456

RESUMO

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/ß-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.


Assuntos
Colo/crescimento & desenvolvimento , Anticorpos de Cadeia Única/metabolismo , Trombospondinas/metabolismo , Via de Sinalização Wnt , Sítios de Ligação , Colo/metabolismo , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/imunologia
11.
Nat Commun ; 11(1): 99, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911617

RESUMO

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.


Assuntos
Melanoma/imunologia , Proteínas Nucleares/imunologia , Linfócitos T Reguladores/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Quimiocina CCL17/genética , Quimiocina CCL17/imunologia , Quimiocina CCL22/genética , Quimiocina CCL22/imunologia , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/imunologia , Humanos , Melanoma/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genética
12.
J Virol ; 94(2)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694946

RESUMO

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways, including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host's IFN-I response, West Nile virus (WNV) replication is sensitive to pretreatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV's sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells, WNV replication is significantly increased and IFN-I induction and signaling are impaired compared to wild-type (wt) cells. IFN-ß pretreatment was more effective in protecting against subsequent WNV infection in wt cells than TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next-generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced JAK1 and STAT1 phosphorylation and impaired induction of several interferon-stimulated genes (ISGs) following WNV infection or IFN-ß treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function are dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identify VAMP8 as a novel regulator of the IFN-I system.IMPORTANCE WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.


Assuntos
Imunidade Inata , Interferon Tipo I/imunologia , Proteínas R-SNARE/imunologia , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , Replicação Viral/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/fisiologia , Células A549 , Deleção de Genes , Células HEK293 , Humanos , Janus Quinase 1/genética , Janus Quinase 1/imunologia , Fosforilação/genética , Fosforilação/imunologia , Proteínas R-SNARE/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Replicação Viral/genética , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/patologia
13.
Am J Respir Cell Mol Biol ; 62(2): 157-167, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31385713

RESUMO

TLR8 (Toll-like receptor 8) is an intracellular pattern recognition receptor that senses RNA in endosomes to initiate innate immune signaling through NF-κB, and mechanisms regulating TLR8 protein abundance are not completely understood. Protein degradation is a cellular process controlling protein concentrations, accomplished largely through ubiquitin transfer directed by E3 ligase proteins to substrates. In the present study, we show that TLR8 has a short half-life in THP-1 monocytes (∼1 h) and that TLR8 is ubiquitinated and degraded in the proteasome. Treatment with the TLR8 agonist R848 causes rapid depletion of TLR8 concentrations at early time points, an effect blocked by proteasomal inhibition. We show a novel role for RNF216 (ring finger protein 216), an E3 ligase that targets TLR8 for ubiquitination and degradation. RNF216 overexpression reduces TLR8 concentrations, whereas RNF216 knockdown stabilizes TLR8. We describe a potential role for TLR8 activation by circulating RNA ligands in humans with acute respiratory distress syndrome (ARDS): Plasma and extracted RNA fractions from subjects with ARDS activated TLR8 in vitro. MicroRNA (miRNA) expression profiling revealed several circulating miRNAs from subjects with ARDS. miRNA mimics promoted TLR8 proteasomal degradation in THP-1 cells. These data show that TLR8 proteasomal disposal through RNF216 in response to RNA ligands regulates TLR8 cellular concentrations and may have implications for innate immune signaling. In addition, TLR8 activation by circulating RNA ligands may be a previously underrecognized stimulus contributing to excessive innate immune signaling characteristic of ARDS.


Assuntos
MicroRNA Circulante/imunologia , Monócitos/metabolismo , Receptor 8 Toll-Like/imunologia , Ubiquitina-Proteína Ligases/imunologia , Proteínas de Transporte/genética , Humanos , Imunidade Inata/imunologia , NF-kappa B/metabolismo , Ubiquitinação/imunologia
14.
J Clin Invest ; 130(3): 1301-1314, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31714898

RESUMO

Influenza A virus (IAV) is among the most common causes of pneumonia-related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection result from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L, and HOIP, is a critical regulator of NF-κB-dependent inflammation. Using mice with lung epithelial-specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial-driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and noninfected cells triggered by the activation of type I IFN receptor and mediated by IRF1, which was maladaptive and contributed to hyperinflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.


Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Pulmão/imunologia , Complexos Multiproteicos/imunologia , Infecções por Orthomyxoviridae/imunologia , Pneumonia Viral/imunologia , Mucosa Respiratória/imunologia , Células A549 , Animais , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia , Pulmão/patologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Pneumonia Viral/genética , Pneumonia Viral/patologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/imunologia
15.
Dev Comp Immunol ; 103: 103501, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31634519

RESUMO

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, participates in both innate and adaptive immunity and regulates the apoptotic process. In this study, we observed that an ortholog of TRAF6 could inhibit the activity of p53 and suppress the apoptotic process in the Hong Kong oyster, Crassostrea hongkongensis. To investigate the possible molecular mechanism of the ChTRAF6-induced antiapoptotic effect, a GST pull-down screening assay was conducted, and ChPellino was found to physically interact with ChTRAF6. In addition, the interaction between them was confirmed by Co-immunoprecipitation. Furthermore, western blotting revealed that the phosphorylation level of ChPellino was decreased after the RNAi of ChTRAF6, demonstrating that ChTRAF6 may be an upstream regulator of Pellino activation. Furthermore, the apoptosis level of hemocytes increased after ChPellino knockdown, and ChPellino overexpression suppressed ChTRAF6-dependent p53 activation. Taken together, these results indicate that ChPellino plays a critical role in suppressing ChTRAF6-dependent anti-apoptosis in the hemocytes of Crassostrea hongkongensis.


Assuntos
Apoptose/imunologia , Crassostrea/imunologia , Hemócitos/imunologia , Fator 6 Associado a Receptor de TNF/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Imunidade Inata/imunologia
16.
Cell Death Dis ; 10(12): 946, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827077

RESUMO

Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor and is involved in the innate immune response against RNA viruses infection. Here, we demonstrate that the Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a positive regulator of the RIG-I-mediated signaling pathway. G3BP1-deficient cells inhibited RNA virus-triggered induction of downstream antiviral genes. Furthermore, we found that G3BP1 inhibited the replication of Sendai virus and vesicular stomatitis virus, indicating a positive regulation of G3BP1 to cellular antiviral responses. Mechanistically, G3BP1 formed a complex with RNF125 and RIG-I, leading to decreased RNF125 via its auto-ubiquitination; thus, promoting expression of RIG-I. Overall, the results suggest a novel mechanism for G3BP1 in the positive regulation of antiviral signaling mediated by RIG-I.


Assuntos
Proteína DEAD-box 58/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Infecções por Vírus de RNA/genética , Ubiquitina-Proteína Ligases/genética , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Vírus de RNA/imunologia , Vírus de RNA/patogenicidade , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/genética , Ubiquitinação/imunologia , Replicação Viral/genética
17.
BMC Plant Biol ; 19(1): 530, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783788

RESUMO

BACKGROUND: Cell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses. RESULTS: Expression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses. CONCLUSION: The results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/imunologia , Oryza/genética , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Xanthomonas/fisiologia , Arabidopsis/imunologia , Parede Celular/imunologia , Oryza/imunologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Proteínas de Plantas/imunologia , Ubiquitina-Proteína Ligases/imunologia , Xanthomonas/enzimologia
18.
Sci Rep ; 9(1): 16862, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727944

RESUMO

Patients with acute myeloid leukemia frequently present translocations of MLL gene. Rearrangements of MLL protein (MLL-r) in complexes that contain the histone methyltransferase DOT1L are common, which elicit abnormal methylation of lysine 79 of histone H3 at MLL target genes. Phase 1 clinical studies with pinometostat (EPZ-5676), an inhibitor of DOT1L activity, demonstrated the therapeutic potential for targeting DOT1L in MLL-r leukemia patients. We previously reported that down-regulation of DOT1L increases influenza and vesicular stomatitis virus replication and decreases the antiviral response. Here we show that DOT1L inhibition also reduces Sendai virus-induced innate response and its overexpression decreases influenza virus multiplication, reinforcing the notion of DOT1L controlling viral replication. Accordingly, genes involved in the host innate response against pathogens (RUBICON, TRIM25, BCL3) are deregulated in human lung epithelial cells treated with pinometostat. Concomitantly, deregulation of some of these genes together with that of the MicroRNA let-7B, may account for the beneficial effects of pinometostat treatment in patients with MLL-r involving DOT1L. These results support a possible increased vulnerability to infection in MLL-r leukemia patients undergoing pinometostat treatment. Close follow up of infection should be considered in pinometostat therapy to reduce some severe side effects during the treatment.


Assuntos
Antineoplásicos/efeitos adversos , Benzimidazóis/efeitos adversos , Inibidores Enzimáticos/efeitos adversos , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Vírus da Influenza A Subtipo H1N1/genética , Infecções Oportunistas/induzido quimicamente , Células A549 , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Proteína 3 do Linfoma de Células B/genética , Proteína 3 do Linfoma de Células B/imunologia , Suscetibilidade a Doenças , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/induzido quimicamente , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , MicroRNAs/imunologia , Infecções Oportunistas/genética , Infecções Oportunistas/imunologia , Infecções Oportunistas/virologia , Vírus Sendai/genética , Vírus Sendai/crescimento & desenvolvimento , Vírus Sendai/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Replicação Viral
19.
J Immunol ; 203(11): 3045-3053, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31611260

RESUMO

Macrophages drive the pathological process of inflammatory bowel diseases (IBD) mostly by secreting proinflammatory cytokines, such as Tnf-α. Recent studies have indicated the association between epigenetic modifications and macrophage functions. However, epigenetic mechanisms regulating macrophages' functional involvement in IBD remain unknown. In this study, we investigated whether the epigenetic regulator Uhrf1 plays a role in innate immunity by functionally regulating macrophages in intestines. We employed two transgenic strains of mice (one with Uhrf1 deficiency in macrophages [Uhrf1fl/flLyz2-Cre mice] and the other with the two mutations at Uhrf1's DNA methylation regulatory site [Uhrf1YP187/188AA mice]) to assess their susceptibility to dextran sodium sulfate-induced colitis. We examined the cytokines derived from Uhrf1fl/flLyz2-Cre and Uhrf1YP187/188AA macrophages in response to LPS stimulation. We also analyzed the effects of proinflammatory cytokines on Uhrf1 expression in macrophages. The data demonstrated that Uhrf1 deficiency and Uhrf1YP187/188AA mutation resulted in severe colitis in the dextran sodium sulfate-treated mice. In vitro analysis revealed the hypomethylation of Tnf-α promoter and the increased Tnf-α expression in Uhrf1fl/flLyz2-Cre and Uhrf1YP187/188AA macrophages in response to LPS stimulation, and anti-Tnf-α therapy implied the key role of Tnf-α to the aggravated colitis in Uhrf1-deficient mice. Exogenous Tnf-α destabilized Uhrf1 protein through ubiquitination-mediated protein degradation, triggering macrophage activation. In conclusion, we identified that Uhrf1-mediated DNA methylation controls Tnf-α expression of macrophages in the experimental colitis resembling IBD. The epigenetic mechanisms that activate macrophages may provide new therapeutic targets for IBD treatment.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Colite/imunologia , Inflamação/imunologia , Doenças Inflamatórias Intestinais/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/deficiência , Proteínas Estimuladoras de Ligação a CCAAT/genética , Modelos Animais de Doenças , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fator de Necrose Tumoral alfa/genética , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética
20.
Front Immunol ; 10: 2147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31620121

RESUMO

Thioredoxin-interacting protein (Txnip) inhibits the activity of thioredoxin (Trx) to modulate inflammatory responses. The burden of inflammation caused by microbial infection is strongly associated with disease severity; however, the role of Txnip in bacterial infection remains unclear. In Group A Streptococcus (GAS)-infected macrophages, Txnip was degraded independent of glucose consumption and streptococcal cysteine protease expression. Treatment with proteasome inhibitors reversed GAS-induced Txnip degradation. The activation of Toll-like receptor 2 (TLR2) initiated Txnip degradation, while no further Txnip degradation was observed in TLR2-deficient bone marrow-derived macrophages. NADPH oxidase-regulated NF-κB activation and pro-inflammatory activation were induced and accompanied by Txnip degradation during GAS infection. Silencing Txnip prompted TLR2-mediated inducible nitric oxide synthase (iNOS)/NO, TNF-α, and IL-6 production whereas the blockage of Txnip degradation by pharmacologically inhibiting the HECT E3 ubiquitin ligase with heclin and AMP-dependent protein kinase with dorsomorphin effectively reduced such effects. Our findings reveal that TLR2/NADPH oxidase-mediated Txnip proteasomal degradation facilitates pro-inflammatory cytokine production during GAS infection.


Assuntos
Proteínas de Transporte/metabolismo , Inflamação/metabolismo , Infecções Estreptocócicas/metabolismo , Tiorredoxinas/metabolismo , Receptor 2 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Transporte/imunologia , Inflamação/imunologia , Camundongos , Células RAW 264.7 , Infecções Estreptocócicas/imunologia , Tiorredoxinas/imunologia , Ubiquitina-Proteína Ligases/imunologia
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