RESUMO
Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.
Assuntos
Neuropilina-2 , Neoplasias de Mama Triplo Negativas , Humanos , Neuropilina-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ligação Proteica , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Neuropilina-1/metabolismoRESUMO
Efzofitimod is a first-in-class biologic based on a naturally occurring splice variant of histidyl-tRNA synthetase (HARS) that downregulates immune responses via selective modulation of neuropilin-2 (NRP2). Preclinical data found high expression of NRP2 in sarcoidosis granulomas. Treatment with efzofitimod reduced the granulomatous inflammation induced by P. acnes in an animal model of sarcoidosis. A dose escalating trial of efzofitimod in sarcoidosis with chronic symptomatic pulmonary disease found that treatment with efzofitimod was associated with improved quality of life with a trend towards reduced glucocorticoid use and stable to improved pulmonary function. These studies have led to a large Phase 3 trial of efzofitimod in symptomatic pulmonary sarcoidosis.
RESUMO
Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability. This compelled us to develop a more convenient high-throughput assay. In this study, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPBMC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs through comparison to known benchmark PS-ASO controls that were previously shown to be safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO TLR9-dependent innate immune activation.
Assuntos
Linfoma de Burkitt , Oligonucleotídeos Antissenso , Humanos , Oligonucleotídeos Antissenso/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/terapia , Leucócitos Mononucleares , Receptor Toll-Like 9/genéticaRESUMO
A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.
Assuntos
Leucócitos Mononucleares , Oligonucleotídeos Antissenso , Humanos , Oligonucleotídeos Antissenso/genética , Voluntários Saudáveis , Interleucina-6/genéticaRESUMO
Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 109/L in 50% and ≥100 × 109/L in 80% of the subjects. However, grade 4 thrombocytopenia (<25 × 109/L) occurred in three subjects in NEURO-TTR trial, and one of these suffered a fatal intracranial hemorrhage. The two others were treated successfully with corticosteroids and discontinuation of inotersen. Investigations in a subset of subjects in NEURO-TTR (n = 17 placebo; n = 31 inotersen) and OLE (n = 33) trials ruled out direct myelotoxicity, consumptive coagulopathy, and heparin-induced thrombocytopenia. Antiplatelet immunoglobulin G (IgG) antibodies were detected at baseline in 5 of 31 (16%) inotersen-treated subjects in NEURO-TTR, 4 of whom eventually developed grade 1 or 2 thrombocytopenia while on the drug. In addition, 24 subjects in the same group developed treatment-emergent antiplatelet IgG antibodies, of which 2 developed grade 2, and 3 developed grade 4 thrombocytopenia. Antiplatelet IgG antibodies in two of the three grade 4 thrombocytopenia subjects targeted GPIIb/IIIa. Plasma cytokines previously implicated in immune dysregulation, such as interleukin (IL)-23 and a proliferation-inducing ligand (APRIL) were often above the normal range at baseline. Collectively, these findings suggest an underlying immunologic dysregulation predisposing some individuals to immune-mediated thrombocytopenia during inotersen treatment.
Assuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Trombocitopenia/sangue , Adulto , Idoso , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/imunologia , Neuropatias Amiloides Familiares/patologia , Feminino , Predisposição Genética para Doença , Humanos , Doenças do Sistema Imunitário/induzido quimicamente , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/patologia , Imunoglobulina G , Hemorragias Intracranianas/induzido quimicamente , Hemorragias Intracranianas/imunologia , Hemorragias Intracranianas/patologia , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/efeitos adversos , Oligonucleotídeos Antissenso/efeitos adversos , Qualidade de Vida , Trombocitopenia/induzido quimicamente , Trombocitopenia/imunologia , Trombocitopenia/patologiaRESUMO
Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.
Assuntos
Imunidade Celular/imunologia , Inflamação/imunologia , Oligonucleotídeos Antissenso/uso terapêutico , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Linfócitos B/imunologia , Citocinas/sangue , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/metabolismo , Cultura Primária de Células , RNA/genética , RNA/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Análise de Célula Única , Receptor Toll-Like 9/genética , Transcrição GênicaRESUMO
The A20 protein has emerged as an important negative regulator of Toll like receptor (TLR) and retinoic acid-inducible gene 1 (RIG-I)-mediated anti-viral signaling. A20 functions both as a RING-type E3 ubiquitin ligase and as a de-ubiquitinating enzyme. Nuclear factor kappa B (NF-kappaB) and interferon regulatory factor (IRF) pathways are targeted by A20 through mechanisms that appear to be both overlapping and distinct, resulting in the downregulation of interferon alpha/beta (IFNalpha/beta) production. This review specifically details the impact of A20 on the cytosolic RIG-I/MDA5 pathway, a process that is less understood than that of NF-kappaB but is essential for the regulation of the innate immune response to viral infection.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Imunidade Inata/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/fisiologia , Vírus/imunologia , Humanos , Interferon-alfa/metabolismo , Interferon beta/metabolismo , NF-kappa B/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , UbiquitinaçãoRESUMO
The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5' triphosphate (5'ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5'pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5'pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5'pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5'pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5'pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , RNA Viral/farmacologia , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Animais , Antivirais/uso terapêutico , Linhagem Celular , Ativação Enzimática , Humanos , Imunidade Inata , Inflamação , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Interferência de RNA , RNA Viral/genética , RNA Viral/metabolismo , RNA Viral/uso terapêutico , Receptores do Ácido Retinoico/genética , Fator de Transcrição STAT1/metabolismo , Transdução de SinaisRESUMO
The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-κB, respectively. Both the IRF and NF-κB pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-κB-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase I-kappa B/metabolismo , Interferons/imunologia , Fator 3 Associado a Receptor de TNF/metabolismo , Estomatite Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Regulação para Baixo , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Camundongos , Camundongos Knockout , Ligação Proteica , Fator 3 Associado a Receptor de TNF/genética , Ubiquitinação , Estomatite Vesicular/genética , Estomatite Vesicular/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase 3-, 7-, and 9-dependent apoptotic activities that functions as an E3 ubiquitin ligase, and it targets caspases for degradation. In this study, we demonstrate that Sendai virus (SeV) infection results in the IKKε- or TBK1-mediated phosphorylation of XIAP in vivo at Ser430, resulting in Lys(48)-linked autoubiquitination at Lys322/328 residues, followed by the subsequent proteasomal degradation of XIAP. Interestingly, IKKε expression and XIAP turnover increases SeV-triggered mitochondrion-dependent apoptosis via the release of caspase 3, whereas TBK1 expression does not increase apoptosis. Interestingly, phosphorylation also regulates XIAP interaction with the transcription factor IRF3, suggesting a role in IRF3-Bax-mediated apoptosis. Our findings reveal a novel function of IKKε as a regulator of the virus-induced triggering of apoptosis via the phosphorylation-dependent turnover of XIAP.
Assuntos
Apoptose , Quinase I-kappa B/metabolismo , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/fisiopatologia , Vírus Sendai/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Humanos , Quinase I-kappa B/genética , Fosforilação , Infecções por Respirovirus/virologia , Vírus Sendai/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
Sensing of RNA virus infection by the RIG-I-like receptors (RLRs) engages a complex signaling cascade that utilizes the mitochondrial antiviral signaling (MAVS) adapter protein to orchestrate the innate host response to pathogen, ultimately leading to the induction of antiviral and inflammatory responses mediated by type I interferon (IFN) and NF-κB pathways. MAVS is localized to the outer mitochondrial membrane, and has been associated with peroxisomes, the endoplasmic reticulum and autophagosomes, where it coordinates signaling events downstream of RLRs. MAVS not only plays a pivotal role in the induction of antiviral and inflammatory pathways but is also involved in the coordination of apoptotic and metabolic functions. This review summarizes recent findings related to the MAVS adapter and its essential role in the innate immune response to RNA viruses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Imunidade Inata , Mitocôndrias/imunologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Transdução de Sinais/imunologia , Fatores de Transcrição/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/imunologia , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon , Mitocôndrias/metabolismo , Membranas Mitocondriais/imunologia , Membranas Mitocondriais/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Peroxissomos/imunologia , Peroxissomos/metabolismo , RNA Helicases/imunologia , RNA Helicases/metabolismo , Infecções por Vírus de RNA/virologia , RNA Viral/imunologia , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-I (RIG-I) results in the activation of signaling cascades that culminate with the generation of the type I interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters, kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKKε is recruited to the C-terminal region of MAVS following Sendai or vesicular stomatitis virus (VSV) infection, mediated by Lys63-linked polyubiquitination of MAVS at Lys500, resulting in inhibition of downstream IFN signaling (Paz et al, Mol Cell Biol, 2009). In this study, we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM), 455-PEENEY-460, within this site is required for TRAF3 binding and activation of IFN antiviral response genes, whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS(-/-) mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication, whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore, recruitment of IKKε to an adjacent C-terminal site (aa 468-540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability, thus contributing to IKKε-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retroalimentação Fisiológica , Imunidade Inata , Interferon Tipo I/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Quinase I-kappa B/metabolismo , Interferon Tipo I/imunologia , Camundongos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Estomatite Vesicular/imunologia , Vesiculovirus/imunologiaAssuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Imunidade Inata , Helicase IFIH1 Induzida por Interferon , Interferons/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , NF-kappa B/metabolismo , RNA Helicases/metabolismo , Receptores Imunológicos , Infecções por Respirovirus/imunologia , Transdução de SinaisRESUMO
The mitochondria-bound adapter MAVS participates in IFN induction by recruitment of downstream partners such as members of the TRAF family, leading to activation of NF-kappaB, and the IRF3 pathways. A yeast two-hybrid search for MAVS-interacting proteins yielded the Polo-box domain (PBD) of the mitotic Polo-like kinase PLK1. We showed that PBD associates with two different domains of MAVS in both dependent and independent phosphorylation events. The phosphodependent association requires the phosphopeptide binding ability of PBD. It takes place downstream of the proline-rich domain of MAVS, within an STP motif, characteristic of the binding of PLK1 to its targets, where the central Thr234 residue is phosphorylated. Its phosphoindependent association takes place at the C terminus of MAVS. PLK1 strongly inhibits the ability of MAVS to activate the IRF3 and NF-kappaB pathways and to induce IFN. Reciprocally, depletion of PLK1 can increase IFN induction in response to RIG-I/SeV or RIG-I/poly(I)-poly(C) treatments. This inhibition is dependent on the phosphoindependent association of PBD at the C terminus of MAVS where it disrupts the association of MAVS with its downstream partner TRAF3. IFN induction was strongly inhibited in cells arrested in G2/M by nocodazole, which provokes increased expression of endogenous PLK1. Interestingly, depletion of PLK1 from these nocodazole-treated cells could restore, at least partially, IFN induction. Altogether, these data demonstrate a new function for PLK1 as a regulator of IFN induction and provide the basis for the development of inhibitors preventing the PLK1/MAVS association to sustain innate immunity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Interferons/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Modelos Biológicos , NF-kappa B/metabolismo , Nocodazol/farmacologia , Fosfopeptídeos/química , Fosforilação , Prolina/química , Estrutura Terciária de Proteína , Treonina/química , Técnicas do Sistema de Duplo-Híbrido , Quinase 1 Polo-LikeRESUMO
Induction of the antiviral interferon response is initiated upon recognition of viral RNA structures by the RIG-I or Mda-5 DEX(D/H) helicases. A complex signaling cascade then converges at the mitochondrial adapter MAVS, culminating in the activation of the IRF and NF-kappaB transcription factors and the induction of interferon gene expression. We have previously shown that MAVS recruits IkappaB kinase epsilon (IKKepsilon) but not TBK-1 to the mitochondria following viral infection. Here we map the interaction of MAVS and IKKepsilon to the C-terminal region of MAVS and demonstrate that this interaction is ubiquitin dependent. MAVS is ubiquitinated following Sendai virus infection, and K63-linked ubiquitination of lysine 500 (K500) of MAVS mediates recruitment of IKKepsilon to the mitochondria. Real-time PCR analysis reveals that a K500R mutant of MAVS increases the mRNA level of several interferon-stimulated genes and correlates with increased NF-kappaB activation. Thus, recruitment of IKKepsilon to the mitochondria upon MAVS K500 ubiquitination plays a modulatory role in the cascade leading to NF-kappaB activation and expression of inflammatory and antiviral genes. These results provide further support for the differential role of IKKepsilon and TBK-1 in the RIG-I/Mda5 pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase I-kappa B/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Células HeLa , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Interferon beta/metabolismo , Lisina/química , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus Sendai/patogenicidade , Transdução de SinaisRESUMO
Intracellular RNA virus infection is detected by the cytoplasmic RNA helicase RIG-I that plays an essential role in signaling to the host antiviral response. Recently, the adapter molecule that links RIG-I sensing of incoming viral RNA to downstream signaling and gene activation events was characterized by four different groups; MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and a carboxyl-terminal mitochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepatitis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its immune evasion strategy. With a novel search program written in python, we also identified an uncharacterized protein, KIAA1271 (K1271), containing a single CARD-like domain at the N terminus and a Leu-Val-rich C terminus that is identical to that of MAVS/IPS-1/VISA/Cardif. Using a combination of biochemical analysis, subcellular fractionation, and confocal microscopy, we now demonstrate that NS3-4A cleavage of MAVS/IPS-1/VISA/Cardif/K1271 results in its dissociation from the mitochondrial membrane and disrupts signaling to the antiviral immune response. Furthermore, virus-induced IKKepsilon kinase, but not TBK1, colocalized strongly with MAVS at the mitochondrial membrane, and the localization of both molecules was disrupted by NS3-4A expression. Mutation of the critical cysteine 508 to alanine was sufficient to maintain mitochondrial localization of MAVS/IPS-1/VISA/Cardif and IKKepsilon in the presence of NS3-4A. These observations provide an outline of the mechanism by which hepatitis C virus evades the interferon antiviral response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hepacivirus , Quinase I-kappa B/metabolismo , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Hepacivirus/química , Humanos , Membranas Intracelulares/metabolismo , Mitocôndrias/ultraestrutura , Peptídeo Hidrolases/metabolismo , Transdução de Sinais/imunologiaRESUMO
Primary effusion lymphoma (PEL) is a herpesvirus-8-associated lymphoproliferative disease characterized by migration of tumor cells to serous body cavities. PEL cells originate from postgerminal center B cells and share a remarkable alteration in B cell transcription factor expression and/or activation with classical Hodgkin's disease cells. Comparative analysis of gene expression by cDNA microarray of BCBL-1 cells (PEL), L-428 (classical Hodgkin's disease), and BJAB cells revealed a subset of genes that were differentially expressed in BCBL-1 cells. Among these, four genes involved in cell migration and chemotaxis were strongly up-regulated in PEL cells: leukotriene A4 (LTA4) hydrolase (LTA4H), IL-16, thrombospondin-1 (TSP-1), and selectin-P ligand (PSGL-1). Up-regulation of LTA4H was investigated at the transcriptional level. Full-length LTA4H promoter exhibited 50% higher activity in BCBL-1 cells than in BJAB or L-428 cells. Deletion analysis of the LTA4H promoter revealed a positive cis-regulatory element active only in BCBL-1 cells in the promoter proximal region located between -76 and -40 bp. Formation of a specific DNA-protein complex in this region was confirmed by EMSA. Coculture of ionophore-stimulated primary neutrophils with BCBL-1 cells leads to an increased production of LTB4 compared with coculture with BJAB and L-428 cells as measured by enzyme immunoassay, demonstrating the functional significance of LTA4H up-regulation.
Assuntos
Epóxido Hidrolases/biossíntese , Epóxido Hidrolases/genética , Leucotrieno B4/biossíntese , Linfoma de Células B/enzimologia , Linfoma de Células B/genética , Regulação para Cima , Linhagem Celular Tumoral , Ativação Enzimática/genética , Epóxido Hidrolases/isolamento & purificação , Epóxido Hidrolases/fisiologia , Perfilação da Expressão Gênica , Doença de Hodgkin/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Interleucina-16/fisiologia , Linfoma de Células B/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Trombospondina 1/biossíntese , Trombospondina 1/genética , Trombospondina 1/fisiologia , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
Intracellular viral infection is detected by the cytoplasmic RNA helicase RIG-I, which has an essential role in initiating the host antiviral response. The adaptor molecule that connects RIG-I sensing of incoming viral RNA to downstream signaling and gene activation has recently been elucidated by four independent research groups, and has been ascribed four different names: MAVS, IPS-1, VISA and Cardif. The fact that MAVS/IPS-1/VISA/Cardif localizes to the mitochondrial membrane suggests a link between viral infection, mitochondrial function and development of innate immunity. Furthermore, the hepatitis C virus NS3/4A protease specifically cleaves MAVS/IPS-1/VISA/Cardif as part of its immune-evasion strategy. These studies highlight a novel role for the mitochondria and for caspase activation and recruitment domain (CARD)-containing proteins in coordinating immune and apoptotic responses.