RESUMO
Adaptation of the functional proteome is essential to counter pathogens during infection, yet precisely timed degradation of these response proteins after pathogen clearance is likewise key to preventing autoimmunity. Interferon regulatory factor 1 (IRF1) plays an essential role as a transcription factor in driving the expression of immune response genes during infection. The striking difference in functional output with other IRFs is that IRF1 also drives the expression of various cell cycle inhibiting factors, making it an important tumor suppressor. Thus, it is critical to regulate the abundance of IRF1 to achieve a 'Goldilocks' zone in which there is sufficient IRF1 to prevent tumorigenesis, yet not too much which could drive excessive immune activation. Using genetic screening, we identified the E3 ligase receptor speckle type BTB/POZ protein (SPOP) to mediate IRF1 proteasomal turnover in human and mouse cells. We identified S/T-rich degrons in IRF1 required for its SPOP MATH domain-dependent turnover. In the absence of SPOP, elevated IRF1 protein levels functionally increased IRF1-dependent cellular responses, underpinning the biological significance of SPOP in curtailing IRF1 protein abundance.
Assuntos
Regulação da Expressão Gênica , Genes Reguladores , Humanos , Animais , Camundongos , Fator Regulador 1 de Interferon/genética , Aclimatação , Fatores ImunológicosRESUMO
Tristetraprolin (TTP) is a critical negative immune regulator. It binds AU-rich elements in the untranslated-regions of many mRNAs encoding pro-inflammatory mediators, thereby accelerating their decay. A key but poorly understood mechanism of TTP regulation is its timely proteolytic removal: TTP is degraded by the proteasome through yet unidentified phosphorylation-controlled drivers. In this study, we set out to identify factors controlling TTP stability. Cellular assays showed that TTP is strongly lysine-ubiquitinated, which is required for its turnover. A genetic screen identified the ubiquitin E3 ligase HUWE1 as a strong regulator of TTP proteasomal degradation, which we found to control TTP stability indirectly by regulating its phosphorylation. Pharmacological assessment of multiple kinases revealed that HUWE1-regulated TTP phosphorylation and stability was independent of the previously characterized effects of MAPK-mediated S52/S178 phosphorylation. HUWE1 function was dependent on phosphatase and E3 ligase binding sites identified in the TTP C-terminus. Our findings indicate that while phosphorylation of S52/S178 is critical for TTP stabilization at earlier times after pro-inflammatory stimulation, phosphorylation of the TTP C-terminus controls its stability at later stages.
Assuntos
Tristetraprolina , Ubiquitina-Proteína Ligases , Fosforilação , Tristetraprolina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Ubiquitina/metabolismo , Estabilidade de RNA/genéticaRESUMO
Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.
Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Proteínas Inibidoras de Apoptose , Proteínas Mitocondriais , Humanos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Microscopia Crioeletrônica , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Ubiquitinação , Multimerização Proteica , Serina Peptidase 2 de Requerimento de Alta Temperatura A/química , Serina Peptidase 2 de Requerimento de Alta Temperatura A/metabolismoRESUMO
Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1-3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Genes myc , Humanos , Masculino , Mitose , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , ProteóliseRESUMO
Lipocalin 2 (LCN2) is a secreted glycoprotein with roles in multiple biological processes. It contributes to host defense by interference with bacterial iron uptake and exerts immunomodulatory functions in various diseases. Here, we aimed to characterize the function of LCN2 in lung macrophages and dendritic cells (DCs) using Lcn2-/- mice. Transcriptome analysis revealed strong LCN2-related effects in CD103+ DCs during homeostasis, with differential regulation of antigen processing and presentation and antiviral immunity pathways. We next validated the relevance of LCN2 in a mouse model of influenza infection, wherein LCN2 protected from excessive weight loss and improved survival. LCN2-deficiency was associated with enlarged mediastinal lymph nodes and increased lung T cell numbers, indicating a dysregulated immune response to influenza infection. Depletion of CD8+ T cells equalized weight loss between WT and Lcn2-/- mice, proving that LCN2 protects from excessive disease morbidity by dampening CD8+ T cell responses. In vivo T cell chimerism and in vitro T cell proliferation assays indicated that improved antigen processing by CD103+ DCs, rather than T cell intrinsic effects of LCN2, contribute to the exacerbated T cell response. Considering the antibacterial potential of LCN2 and that commensal microbes can modulate antiviral immune responses, we speculated that LCN2 might cause the observed influenza phenotype via the microbiome. Comparing the lung and gut microbiome of WT and Lcn2-/- mice by 16S rRNA gene sequencing, we observed profound effects of LCN2 on gut microbial composition. Interestingly, antibiotic treatment or co-housing of WT and Lcn2-/- mice prior to influenza infection equalized lung CD8+ T cell counts, suggesting that the LCN2-related effects are mediated by the microbiome. In summary, our results highlight a novel regulatory function of LCN2 in the modulation of antiviral immunity.
Assuntos
Influenza Humana/imunologia , Lipocalina-2/metabolismo , Microbiota/imunologia , Transcriptoma , Animais , Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Microbioma Gastrointestinal , Homeostase , Humanos , Imunidade , Influenza Humana/virologia , Lipocalina-2/genética , Pulmão/imunologia , Pulmão/virologia , Ativação Linfocitária , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos EspecíficosRESUMO
The rapid and dynamic activation of the innate immune system is achieved through complex signaling networks regulated by post-translational modifications modulating the subcellular localization, activity, and abundance of signaling molecules. Many constitutively expressed signaling molecules are present in the cell in inactive forms, and become functionally activated once they are modified with ubiquitin, and, in turn, inactivated by removal of the same post-translational mark. Moreover, upon infection resolution a rapid remodeling of the proteome needs to occur, ensuring the removal of induced response proteins to prevent hyperactivation. This review discusses the current knowledge on the negative regulation of innate immune signaling pathways by deubiquitinating enzymes, and through degradative ubiquitination. It focusses on spatiotemporal regulation of deubiquitinase and E3 ligase activities, mechanisms for re-establishing proteostasis, and degradation through immune-specific feedback mechanisms vs. general protein quality control pathways.
Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação/fisiologia , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Ubiquitinação/genética , Ubiquitinação/imunologiaRESUMO
Tripartite motif protein 52 (TRIM52) is a non-canonical TRIM family member harbouring the largest RING domain encoded in the human genome. In humans TRIM52 is conserved and has been under positive selection pressure, yet it has been lost in many non-primates. Competitive cell fitness assays demonstrated that TRIM52 ablation reduces cellular fitness in multiple different cell types. To better understand how this cell-essential factor is controlled, we investigated how expression of this non-canonical protein is regulated. Here, we show that TRIM52 mRNA is constitutively expressed from an intergenic region preceding the TRIM52 gene. Yet, TRIM52 protein is rapidly turned-over by the proteasome with a 3.5-minute half-life, one of the shortest in the human proteome. Consistent with this extremely rapid degradation rate, all three TRIM52 domains were identified to contribute to its instability. Intriguingly, a repetitive acidic loop in the RING domain was identified as one of the main destabilizing regions, which was unexpected given the prevailing notion that these sequences are poor proteasome substrates. This work indicates that the effect of such repetitive acidic regions on proteasomal degradation depends on the protein context, and it identifies TRIM52 as an attractive model protein to study what these contextual properties are.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Sequências Repetitivas de Aminoácidos , Proteínas com Motivo Tripartido/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HEK293 , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA-Seq , Análise de Célula Única , Fatores de Tempo , Proteínas com Motivo Tripartido/químicaRESUMO
Vunjak and Versteeg introduce the TRIM family of post-translational modifiers and the roles of these proteins in viral restriction, immune signaling and autophagy.
Assuntos
Autofagia , Imunidade Inata/genética , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido/fisiologia , Viroses/imunologia , Animais , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/imunologiaRESUMO
Tripartite motif (TRIM) proteins have been shown to play important roles in cancer development and progression by modulating cell proliferation or resistance from cell death during non-homeostatic stress conditions found in tumor micro-environments. In this study, we set out to investigate the importance for cellular fitness of the virtually uncharacterized family member TRIM52. The human TRIM52 gene has arisen recently in evolution, making it unlikely that TRIM52 is required for basic cellular functions in normal cells. However, a recent genome-wide ablation screening study has suggested that TRIM52 may be essential for optimal proliferation or survival in certain genetic cancer backgrounds. Identifying genes which fit this concept of genetic context-dependent fitness in cancer cells is of interest as they are promising targets for tumor-specific therapy. We report here that TRIM52 ablation significantly diminished the proliferation of specific glioblastoma cell lines in cell culture and mouse xenografts by compromising their cell cycle progression in a p53-dependent manner. Together, our findings point to a non-redundant TRIM52 function that is required for optimal proliferation.
RESUMO
Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.
Assuntos
Linfócitos B/imunologia , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Humanos , Transdução de Sinais , UbiquitinaçãoRESUMO
The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The ß-transducin repeat-containing protein (ß-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. ß-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation. Here we report that TRIM9 is a negative regulator of NF-κB activation. Interaction between the phosphorylated degron motif of TRIM9 and the WD40 repeat region of ß-TrCP prevented ß-TrCP from binding its substrates, stabilizing IκBα and p100 and thereby blocking NF-κB activation. Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production. This study not only elucidates a mechanism for TRIM9-mediated regulation of the ß-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.
Assuntos
Encéfalo/metabolismo , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Microscopia Confocal , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Proteínas com Motivo TripartidoRESUMO
During the immune response, striking the right balance between positive and negative regulation is critical to effectively mount an anti-microbial defense while preventing detrimental effects from exacerbated immune activation. Intra-cellular immune signaling is tightly regulated by various post-translational modifications, which allow for this dynamic response. One of the post-translational modifiers critical for immune control is ubiquitin, which can be covalently conjugated to lysines in target molecules, thereby altering their functional properties. This is achieved in a process involving E3 ligases which determine ubiquitination target specificity. One of the most prominent E3 ligase families is that of the tripartite motif (TRIM) proteins, which counts over 70 members in humans. Over the last years, various studies have contributed to the notion that many members of this protein family are important immune regulators. Recent studies into the mechanisms by which some of the TRIMs regulate the innate immune system have uncovered important immune regulatory roles of both covalently attached, as well as unanchored poly-ubiquitin chains. This review highlights TRIM evolution, recent findings in TRIM-mediated immune regulation, and provides an outlook to current research hurdles and future directions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Imunidade Inata , Transdução de Sinais , Motivos de Aminoácidos , Animais , Citocinas/imunologia , Evolução Molecular , Humanos , Invertebrados/classificação , Proteínas de Membrana/imunologia , Ubiquitina/imunologiaRESUMO
Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.
Assuntos
Quinase I-kappa B/imunologia , Interferon Tipo I/imunologia , Poliubiquitina/biossíntese , Ubiquitina-Proteína Ligases/imunologia , Animais , Antivirais , Células Cultivadas , Ativação Enzimática/imunologia , Humanos , Janus Quinase 1 , Camundongos , Fosforilação/imunologia , Interferência de RNA , RNA Interferente Pequeno , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Tripartite motif (TRIM) proteins have been implicated in multiple cellular functions, including antiviral activity. Research efforts so far indicate that the antiviral activity of TRIMs relies, for the most part, on their function as E3-ubiquitin ligases. A substantial number of the TRIM family members have been demonstrated to mediate innate immune cell signal transduction and subsequent cytokine induction. In addition, a subset of TRIMs has been shown to restrict viral replication by directly targeting viral proteins. Although the body of work on the cellular roles of TRIM E3-ubiquitin ligases has rapidly grown over the last years, many aspects of their molecular workings and multi-functionality remain unclear. The antiviral function of many TRIMs seems to be conferred by specific isoforms, by sub-cellular localization and in cell-type-specific contexts. Here we review recent findings on TRIM antiviral functions, current limitations and an outlook for future research.
Assuntos
Antivirais/uso terapêutico , Imunidade Inata/imunologia , Ubiquitina-Proteína Ligases/imunologia , Viroses/tratamento farmacológico , Replicação Viral/imunologia , Vírus/imunologia , Animais , Humanos , Viroses/imunologia , Viroses/virologiaRESUMO
Innate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs. We demonstrate that roughly half of the 75 TRIM-family members enhanced the innate immune response and that they do this at multiple levels in signaling pathways. Moreover, messenger RNA levels and localization of most of these TRIMs were found to be altered during viral infection, suggesting that their regulatory activities are highly controlled at both pre- and posttranscriptional levels. Taken together, our data demonstrate a very considerable dedication of this large protein family to the positive regulation of the antiviral response, which supports the notion that this family of proteins evolved as a component of innate immunity.
Assuntos
Proteínas de Transporte/genética , Imunidade Inata , Leucócitos Mononucleares/metabolismo , RNA Mensageiro/genética , Receptores de Reconhecimento de Padrão/genética , Infecções por Rhabdoviridae/metabolismo , Dedos de Zinco/genética , Processamento Alternativo , Fatores de Restrição Antivirais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/imunologia , Linhagem Celular , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , RNA Mensageiro/imunologia , RNA Interferente Pequeno/genética , Receptores de Reconhecimento de Padrão/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Transdução de Sinais , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Vesiculovirus/imunologia , Dedos de Zinco/imunologiaRESUMO
Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-ß in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.
Assuntos
RNA Helicases DEAD-box/metabolismo , Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Interferons/biossíntese , Ubiquitinação , Proteínas não Estruturais Virais/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Cães , Células HeLa , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Interferons/genética , Camundongos , Camundongos Knockout , Receptores Imunológicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Células Vero , Proteínas não Estruturais Virais/genéticaRESUMO
Detection of invading pathogens by pattern recognition receptors (PRRs) is crucial for the activation of the innate immune response. These sensors signal through intertwining signaling cascades which result in the expression of pro-inflammatory cytokines and type I interferons. Conjugation, or binding, of ubiquitin and ubiquitin-like modifiers (UBLs) to a plethora of immune signaling molecules forms a common theme in innate immune regulation. Numerous E3 ligases and deubiquitylating enzymes (DUBs) actively modify signaling components in order to achieve a balanced activation of the innate immune system. This review will discuss how this balance is achieved and which questions remain regarding innate immune regulation by ubiquitin and UBLs.
Assuntos
Imunidade Inata/imunologia , Ubiquitina/imunologia , Animais , Humanos , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear. One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6, can mediate ISGylation.Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15 conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over expression enhanced the IFNß promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells, suggesting that HerC6(-/-) mice may provide a feasible model to study the role of human HerC5 in antiviral responses.
Assuntos
Citocinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/genética , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Interferon beta/genética , Interferon beta/metabolismo , Interferon beta/farmacologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Células NIH 3T3 , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genéticaRESUMO
A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.
Assuntos
Antivirais/farmacologia , Naftalimidas/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Vesiculovirus/efeitos dos fármacos , Animais , Antivirais/química , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Naftalimidas/química , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/deficiência , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Type I interferons (IFNs) play a crucial role in the innate immune avant-garde against viral infections. Virtually all viruses have developed means to counteract the induction, signaling, or antiviral actions of the IFN circuit. Over 170 different virus-encoded IFN antagonists from 93 distinct viruses have been described up to now, indicating that most viruses interfere with multiple stages of the IFN response. Although every viral IFN antagonist is unique in its own right, four main mechanisms are employed to circumvent innate immune responses: (i) general inhibition of cellular gene expression, (ii) sequestration of molecules in the IFN circuit, (iii) proteolytic cleavage, and (iv) proteasomal degradation of key components of the IFN system. The increasing understanding of how different viral IFN antagonists function has been translated to the generation of viruses with mutant IFN antagonists as potential live vaccine candidates. Moreover, IFN antagonists are attractive targets for inhibition by small-molecule compounds.