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Cancer stem cells (CSCs) actively reprogram their tumor microenvironment (TME) to sustain a supportive niche, which may have a dramatic impact on prognosis and immunotherapy. However, our knowledge of the landscape of the gastric cancer stem-like cell (GCSC) microenvironment needs to be further improved. A multi-step process of machine learning approaches was performed to develop and validate the prognostic and predictive potential of the GCSC-related score (GCScore). The high GCScore subgroup was not only associated with stem cell characteristics, but also with a potential immune escape mechanism. Furthermore, we experimentally demonstrated the upregulated infiltration of CD206+ tumor-associated macrophages (TAMs) in the invasive margin region, which in turn maintained the stem cell properties of tumor cells. Finally, we proposed that the GCScore showed a robust capacity for prediction for immunotherapy, and investigated potential therapeutic targets and compounds for patients with a high GCScore. The results indicate that the proposed GCScore can be a promising predictor of prognosis and responses to immunotherapy, which provides new strategies for the precision treatment of GCSCs.
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Neoplasias Gástricas , Humanos , Imunoterapia/métodos , Células-Tronco Neoplásicas/patologia , Farmacogenética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Microambiente TumoralRESUMO
Time-series Interferometric Synthetic Aperture Radar (InSAR) technology, renowned for its high-precision, wide coverage, and all-weather capabilities, has become an essential tool for Earth observation. However, the quality of the interferometric baseline network significantly influences the monitoring accuracy of InSAR technology. Therefore, optimizing the interferometric baseline is crucial for enhancing InSAR's monitoring accuracy. Surface vegetation changes can disrupt the coherence between SAR images, introducing incoherent noise into interferograms and reducing InSAR's monitoring accuracy. To address this issue, we propose and validate an optimization method for the InSAR baseline that considers changes in vegetation coverage (OM-InSAR-BCCVC) in the Yuanmou dry-hot valley. Initially, based on the imaging times of SAR image pairs, we categorize all interferometric image pairs into those captured during months of high vegetation coverage and those from months of low vegetation coverage. We then remove the image pairs with coherence coefficients below the category average. Using the Small Baseline Subset InSAR (SBAS-InSAR) technique, we retrieve surface deformation information in the Yuanmou dry-hot valley. Landslide identification is subsequently verified using optical remote sensing images. The results show that significant seasonal changes in vegetation coverage in the Yuanmou dry-hot valley lead to noticeable seasonal variations in InSAR coherence, with the lowest coherence in July, August, and September, and the highest in January, February, and December. The average coherence threshold method is limited in this context, resulting in discontinuities in the interferometric baseline network. Compared with methods without baseline optimization, the interferometric map ratio improved by 17.5% overall after applying the OM-InSAR-BCCVC method, and the overall inversion error RMSE decreased by 0.5 rad. From January 2021 to May 2023, the radar line of sight (LOS) surface deformation rate in the Yuanmou dry-hot valley, obtained after atmospheric correction by GACOS, baseline optimization, and geometric distortion region masking, ranged from -73.87 mm/year to 127.35 mm/year. We identified fifteen landslides and potential landslide sites, primarily located in the northern part of the Yuanmou dry-hot valley, with maximum subsidence exceeding 100 mm at two notable points. The OM-InSAR-BCCVC method effectively reduces incoherent noise caused by vegetation coverage changes, thereby improving the monitoring accuracy of InSAR.
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PURPOSE: Immune checkpoint inhibitors (ICIs) have shown durable responses in various malignancies. However, the response to ICI therapy is unpredictable, and investigation of predictive biomarkers needs to be improved. EXPERIMENTAL DESIGN: In total, 120 patients receiving ICI therapy and 40 patients receiving non-ICI therapy were enrolled. Peripheral blood immune cell markers (PBIMs), as liquid biopsy biomarkers, were analyzed by flow cytometry before ICI therapy, and before the first evaluation. In the ICI cohort, patients were randomly divided into training (n = 91) and validation (n = 29) cohorts. Machine learning algorithms were applied to construct the prognostic and predictive immune-related models. RESULTS: Using the training cohort, a peripheral blood immune cell-based signature (BICS) based on four hub PBIMs was developed. In both the training and the validation cohorts, and the whole cohort, the BICS achieved a high accuracy for predicting overall survival (OS) benefit. The high-BICS group had significantly shorter progression-free survival and OS than the low-BICS group. The BICS demonstrated the predictive ability of patients to achieve durable clinical outcomes. By integrating these PBIMs, we further constructed and validated the support vector machine-recursive and feature elimination classifier model, which robustly predicts patients who will achieve optimal clinical benefit. CONCLUSIONS: Dynamic PBIM-based monitoring as a noninvasive, cost-effective, highly specific and sensitive biomarker has broad potential for prognostic and predictive utility in patients receiving ICI therapy.
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Inibidores de Checkpoint Imunológico , Neoplasias , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/tratamento farmacológico , Algoritmos , Citometria de Fluxo , Biópsia LíquidaRESUMO
RIG-I and MDA5 are cytoplasmic RNA sensors that mediate cell intrinsic immunity against viral pathogens. While it has been well-established that RIG-I and MDA5 recognize RNA viruses, their interactive network with DNA viruses, including herpes simplex virus 1 (HSV-1), remains less clear. Using a combination of RNA-deep sequencing and genetic studies, we show that the γ134.5 gene product, a virus-encoded virulence factor, enables HSV growth by neutralization of RIG-I dependent restriction. When expressed in mammalian cells, HSV-1 γ134.5 targets RIG-I, which cripples cytosolic RNA sensing and subsequently suppresses antiviral gene expression. Rather than inhibition of RIG-I K63-linked ubiquitination, the γ134.5 protein precludes the assembly of RIG-I and cellular chaperone 14-3-3ε into an active complex for mitochondrial translocation. The γ134.5-mediated inhibition of RIG-I-14-3-3ε binding abrogates the access of RIG-I to mitochondrial antiviral-signaling protein (MAVS) and activation of interferon regulatory factor 3. As such, unlike wild type virus HSV-1, a recombinant HSV-1 in which γ134.5 is deleted elicits efficient cytokine induction and replicates poorly, while genetic ablation of RIG-I expression, but not of MDA5 expression, rescues viral growth. Collectively, these findings suggest that viral suppression of cytosolic RNA sensing is a key determinant in the evolutionary arms race of a large DNA virus and its host.
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Proteína DEAD-box 58/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/patogenicidade , Receptores Imunológicos/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Animais , Chlorocebus aethiops , Fibroblastos/metabolismo , Células HEK293 , Herpesvirus Humano 1/metabolismo , Humanos , Mitocôndrias/metabolismo , Transporte Proteico/fisiologia , Células VeroRESUMO
Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.
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Linfoma de Burkitt/patologia , Citocromos b/genética , Regulação Neoplásica da Expressão Gênica , Lisossomos/patologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfoma de Burkitt/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Infecções por Vírus Epstein-Barr/complicações , FMN Redutase/genética , Células HEK293 , Herpesvirus Humano 4/isolamento & purificação , Humanos , Lisossomos/genética , Mitocôndrias/genética , Mitocôndrias/patologiaRESUMO
BACKGROUND: Anlotinib, a tyrosine kinase inhibitor, has shown encouraging anti-tumor activity in esophageal squamous cell carcinoma (ESCC). This study was designed to assess the efficacy and safety of anlotinib plus paclitaxel and cisplatin (TP) as first-line therapy for advanced ESCC. METHODS: In a multi-center, single-arm, phase II clinical trial, patients (aged > 18 years) with ESCC, which was judged to be locally advanced, recurrent, or metastatic, received 10 mg oral anlotinib once daily on days 1-14, 135 mg/m2 intravenous paclitaxel on day 1, and 60-75 mg/m2 intravenous cisplatin on days 1-3 every 3 weeks for a maximum of 4-6 cycles as the initial therapy in five centers in China. Subsequently, patients received anlotinib monotherapy (10 mg) as maintenance therapy until tumor progression or intolerable toxicity. The primary endpoint was progression-free survival (PFS). RESULTS: Forty-seven patients were enrolled in this study between October 2019 and March 2021. The median follow-up was 14.04 months (IQR, 9.30-19.38). Of 46 with assessable efficacy, the median PFS and median overall survival were 8.38 months (95% CI, 6.59-10.17) and 18.53 months (95% CI, 13.11-23.95), respectively. The objective response rate was 76.1% (95% CI, 61.2-87.4%), with 4 (8.7%) complete responses and 31 (67.4%) partial responses. The disease control rate was 91.3% (95% CI, 79.2-97.6%). The median duration of response was 6.80 months (95% CI, 4.52-9.08), and 1 patient had an ongoing response for 23 months. Subgroup analysis revealed no association between clinical factors and survival or response. Of the 47 patients with assessable safety, the main grade ≥ 3 treatment-emergent adverse events (TEAEs) were neutropenia (17.0%), bone marrow suppression (12.8%), and vomiting (10.6%). No treatment-related deaths or serious TEAEs were observed. Notably, higher c-Kit levels were an independent factor for superior PFS (HR = 0.032; 95% CI, 0.002-0.606; P = 0.022). CONCLUSIONS: The study demonstrated a manageable safety profile and durable clinical response of anlotinib plus TP as first-line therapy in advanced ESCC, which suggested a potential therapeutic option for this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT04063683. Registered 21 August 2019.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Paclitaxel/efeitos adversos , Cisplatino/efeitos adversos , Neoplasias Esofágicas/tratamento farmacológico , ChinaRESUMO
Pseudorabies virus (PRV) infection could cause severe histopathological damage via releasing multiple factors, including cytokines, peptides, etc. Here, peptidomic results showed that 129 peptides were identified in PRV-infected mouse lungs and were highly involved in the process of PRV infection. The role of one down-regulated biological peptide (designated as AGDP) during PRV infection was investigated. To verify the expression profiles of AGDP in response to PRV infection, the expression level of the precursor protein of AGDP mRNA was significantly decreased in PRV-infected mouse lungs and cells. The synthesized AGDP-treating cells were less susceptible to PRV challenges than the controls, as demonstrated by the decreased virus production and gE expression. AGDP not only inhibited the expression of TNF-α and IL-8 but also appeared to suppress the extracellular release of high-mobility group box 1 (HMGB1) by inhibiting the output of nuclear HMGB1 in cells. AGDP could also inhibit the degradation of IκBα and the phosphorylation levels of P65 after PRV infection. In total, our results revealed many meaningful peptides involved in PRV infection, thereby enhancing the current understanding of the host response to PRV infection, and how AGDP may serve as a promising candidate for developing novel anti-PRV drugs.
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Proteína HMGB1 , Herpesvirus Suídeo 1 , Pseudorraiva , Animais , Citocinas , Pulmão/patologia , Camundongos , Pseudorraiva/tratamento farmacológicoRESUMO
Metastasis is the main event leading to death in cancer patients. Over the past decade, high-throughput technologies have provided genome-wide view of transcriptomic changes associated with cancer metastases. Many microarray and RNA sequencing studies have addressed metastases-related expression patterns in various types of cancer, and the number of relevant works continues to increase rapidly. These works have characterized genes that orchestrate the metastatic phenotype of cancer cells. However, these expression data have been deposited in various repositories, and efficiently analyzing these data is still difficult because of the lack of an integrated data mining platform. To facilitate the in-depth analyses of transcriptome data on metastasis, it is quite important to make a comprehensive integration of these metastases-related expression data. Here, we presented a database, HCMDB (the human cancer metastasis database, http://hcmdb.i-sanger.com/index), which is freely accessible to the research community query cross-platform transcriptome data on metastases. HCMDB is developed and maintained as a useful resource for building the systems-biology understanding of metastasis.
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Bases de Dados Genéticas , Metástase Neoplásica/genética , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Transcriptoma , Coleta de Dados , Apresentação de Dados , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Análise de Sequência de RNA , Interface Usuário-ComputadorRESUMO
Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.
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Regulação Neoplásica da Expressão Gênica/imunologia , Regulação Viral da Expressão Gênica/imunologia , Herpesvirus Humano 4/imunologia , Doença de Hodgkin/imunologia , Linfoma de Células B/imunologia , Neoplasias Experimentais/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Centro Germinativo/imunologia , Centro Germinativo/patologia , Herpesvirus Humano 4/genética , Doença de Hodgkin/genética , Doença de Hodgkin/patologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos , Camundongos Mutantes , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Proteínas da Matriz Viral/genéticaRESUMO
The γ134.5 gene of herpes simplex virus 1 (HSV-1) encodes a virulence factor that promotes viral pathogenesis. Although it perturbs TANK-binding kinase 1 (TBK1) in the complex network of innate immune pathways, the underlying mechanism is obscure. Here we report that HSV-1 γ134.5 targets stimulator of interferon genes (STING) in the intracellular DNA recognition pathway that regulates TBK1 activation. In virus-infected cells the γ134.5 protein associates with and inactivates STING, which leads to downregulation of interferon regulatory factor 3 (IRF3) and IFN responses. Importantly, HSV-1 γ134.5 disrupts translocation of STING from the endoplasmic reticulum to Golgi apparatus, a process necessary to prime cellular immunity. Deletion of γ134.5 or its amino-terminal domain from HSV-1 abolishes the observed inhibitory activities. Consistently, an HSV mutant that lacks functional γ134.5 replicated less efficiently in STING+/+ than in STING-/- mouse embryonic fibroblasts. Moreover, reconstituted expression of human STING in the STING-/- cells activated IRF3 and reduced viral growth. These results suggest that control of the DNA sensing pathway by γ134.5 is advantageous to HSV infection.IMPORTANCE Viral inhibition of innate immunity contributes to herpes simplex virus pathogenesis. Although this complex process involves multiple factors, the underlying events remain unclear. We demonstrate that an HSV virulence factor γ134.5 precludes the activation of STING, a central adaptor in the intracellular DNA sensing pathway. Upon HSV infection, this viral protein engages with and inactivates STING. Consequently, it compromises host immunity and facilitates HSV replication. These observations uncover an HSV mechanism that is likely to mediate viral virulence.
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Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Proteínas de Membrana/antagonistas & inibidores , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Replicação Viral , Animais , Linhagem Celular , Chlorocebus aethiops , Regulação para Baixo , Deleção de Genes , Teste de Complementação Genética , Herpesvirus Humano 1/imunologia , Humanos , Fator Regulador 3 de Interferon/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Virais/genética , Fatores de Virulência/genéticaRESUMO
The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase, RIG-I, and MDA-5. However, its precise role is incompletely defined. By screening a human cDNA library, we showed that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes TBK1 from access to Hsp90 and interferon (IFN) promoter activation. Consistently, the Us11 protein, upon HSV infection, suppresses the expression of beta interferon (IFN-ß), RANTES, and interferon-stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome-dependent pathway. Accordingly, Us11 expression facilitates HSV growth. In contrast, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection.IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrated that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions.
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Proteínas de Choque Térmico HSP90/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Animais , Chlorocebus aethiops , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Células HEK293 , Herpes Simples/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Fosforilação , Ligação Proteica , Transdução de Sinais , Células VeroRESUMO
Like other enteroviruses, enterovirus 71 (EV71) relies on phosphatidylinositol 4-kinase IIIß (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor, ACBD3, is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3, and 3A are all localized to the viral-RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by small interfering RNA (siRNA) leads to a reduction in PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites.IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme, phosphatidylinositol 4-kinase IIIß, by interacting with ACBD3, which alters cellular membranes through the production of a lipid, PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide new insight into the molecular network of enterovirus replication.
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Epstein-Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell-specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.
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Centro Germinativo/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Autoanticorpos/química , Doenças Autoimunes/metabolismo , Doenças Autoimunes/virologia , Diferenciação Celular , Linhagem da Célula , Cruzamentos Genéticos , Epigênese Genética , Citometria de Fluxo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Imunidade Humoral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Baço/citologia , Dedos de ZincoRESUMO
The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.
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Linfócitos B/metabolismo , Transformação Celular Viral , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/metabolismo , Fator 1 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Proteínas da Matriz Viral/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Herpesvirus Humano 4/imunologia , Humanos , Lisina/metabolismo , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator 1 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/antagonistas & inibidores , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Innate immunity provides the first line of host defense against invading microbial pathogens. This defense involves retinoic acid-inducible gene-I-like receptors that detect viral RNA and activate the mitochondrial antiviral-signaling (MAVS) protein, an adaptor protein, leading to activation of the innate antiviral immune response. The mechanisms by which the MAVS signalosome assembles on mitochondria are only partially understood. Here, we identify tripartite motif 14 (TRIM14) as a mediator in the immune response against viral infection. TRIM14 localizes to the outer membrane of mitochondria and interacts with MAVS. Upon viral infection, TRIM14 undergoes Lys-63-linked polyubiquitination at Lys-365 and recruits NF-κB essential modulator to the MAVS signalosome, leading to the activation of both the IFN regulatory factor 3 and NF-κB pathways. Knockdown of TRIM14 disrupts the association between NF-κB essential modulator and MAVS and attenuates the antiviral response. Our results indicate that TRIM14 is a component of the mitochondrial antiviral immunity that facilitates the immune response mediated by retinoic acid-inducible gene-I-like receptors.
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Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas de Transporte/imunologia , Imunidade Inata/genética , Complexos Multiproteicos/metabolismo , Transdução de Sinais/imunologia , Tretinoína/metabolismo , Viroses/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Cromatografia em Gel , Primers do DNA/genética , Humanos , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia de Fluorescência , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Proteínas com Motivo TripartidoRESUMO
As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas Virais/metabolismo , Animais , Chlorocebus aethiops , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Mapeamento de Interação de Proteínas , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Vero , Liberação de Vírus , Receptor de Lamina BRESUMO
The harmful effects of perfluorooctane sulfonate (PFOS) are of growing international concern. This paper aimed to gain an integrated understanding of fitness-related ecological end points, such as behavior, metabolism and swimming physiology, in juvenile Spinibarbus sinensis in response to PFOS toxicity at different temperatures. The fish were exposed to a range of PFOS concentrations (0, 0.32, 0.8, 2 and 5 mg/L) at different temperatures (18 and 28 °C) for 30 days. The effects on fish behavior, metabolic characteristics and aerobic swimming performance caused by PFOS at different temperatures were investigated. Our results showed that both PFOS and temperature had important influences on spontaneous swimming behavior, social interactions, routine metabolic rate (RMR), net energetic cost of transport (COTnet) and critical swimming speed (U crit) in fish. The lowest observed effect concentration for both U crit and RMR was 5 and 0.8 mg/L at 18 and 28 °C, respectively. We found that PFOS affected various behavioral and social end points and also appeared to affect metabolic rates and reduced U crit, likely as a result of increased COTnet, and that many of these effects also changed with respect to temperature. Our results further the understanding of the metabolic and behavioral toxicity of PFOS to aquatic organisms.
Assuntos
Ácidos Alcanossulfônicos/toxicidade , Comportamento Animal/efeitos dos fármacos , Cyprinidae/fisiologia , Metabolismo Energético/efeitos dos fármacos , Fluorocarbonos/toxicidade , Natação/fisiologia , Poluentes Químicos da Água/toxicidade , Animais , Metabolismo Energético/fisiologia , TemperaturaRESUMO
BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-ß/δ is a transcription factor that belongs to the nuclear hormone receptor family. There is little information about the effects of the immediate administration of specific ligands of PPAR-ß/δ (GW0742) in animal models of acute ischemic stroke. Using a rat model of middle cerebral ischemia occlusion (MCAO) in vivo, we have investigated the effect of pretreatment with GW0742 before MCAO. METHODS: The neuroprotective effect of GW0742 against acute ischemic stroke was evaluated by the neurologic deficit score (NDS), dry-wet weight, and 2,3,5-triphenyltetrazolium chloride staining. The levels of interleukin (IL)-1ß, nuclear factor (NF)-κB, and tumor necrosis factor (TNF)-α were detected by an enzyme-linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were detected by Western blot. The apoptotic cells were counted by in situ terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. RESULTS: The pretreatment with GW0742 significantly increased the expression of Bcl-2, and significantly decreased in the volume of infarction, NDS, edema, expressions of IL-1ß, NF-κB, TNFα, and Bax, contents of iNOS and the apoptotic cells in infarct cerebral hemisphere compared with rats in the vehicle group at 24 hours after MCAO. CONCLUSIONS: The study suggests the neuroprotective effect of the PPAR-ß/δ ligand GW0742 in acute ischemic stroke by a mechanism that may involve its anti-inflammatory and antiapoptotic action.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , PPAR delta/agonistas , PPAR beta/agonistas , Acidente Vascular Cerebral/tratamento farmacológico , Tiazóis/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Interleucina-1beta/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/fisiopatologia , Tiazóis/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nitroxyl (HNO) is an important reactive nitrogen that is associated with various states in physiology and pathology and plays a unique function in living systems. So, it is important to exploit fluorescent probes with high sensitivity and selectivity for sensing HNO. In this paper, a novel ratiometric fluorescent probe for HNO was developed utilizing intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) mechanisms. The probe selected coumarin as energy donor, naphthalimide as energy receptor and 2-(diphenylphosphino)benzoate as the sensing site for detecting HNO. When HNO was not present, the 2-(diphenylphosphino)benzoate unit of the probe restricted electron transfer and the ICT process could not occur, leading to the inhibition of FRET process as well. Thus, in the absence of HNO the probe displayed the intrinsic blue fluorescence of coumarin. When HNO was added, the HNO reacted with the 2-(diphenylphosphino)benzoate unit of the probe to yield a hydroxyl group which resulting in the opening of ICT process and the occurring of FRET process. Thus, after providing HNO the probe displayed yellow fluorescence. In addition, the probe showed good linearity in the ratio of fluorescence intensity at 545 nm and 472 nm (I545 nm/I472 nm) with a concentration of HNO (0.1-20 µM). The probe processed a detection limit of 0.014 µM and a response time of 4 min. The probe also specifically identified HNO over a wide pH scope (pH = 4.00-10.00), including physiological conditions. Cellular experiments had shown that this fluorescent probe was virtually non-cytotoxic and could be applied for ratiometric sensing of HNO in A549 cells.
RESUMO
Physiological regulation of transgene expression is a major challenge in gene therapy. Onasemnogene abeparvovec (Zolgensma®) is an approved adeno-associated virus (AAV) vector gene therapy for infants with spinal muscular atrophy (SMA), however, adverse events have been observed in both animals and patients following treatment. The construct contains a native human survival motor neuron 1 (hSMN1) transgene driven by a strong, cytomegalovirus enhancer/chicken ß-actin (CMVen/CB) promoter providing high, ubiquitous tissue expression of SMN. We developed a second-generation AAV9 gene therapy expressing a codon-optimized hSMN1 transgene driven by a promoter derived from the native hSMN1 gene. This vector restored SMN expression close to physiological levels in the central nervous system and major systemic organs of a severe SMA mouse model. In a head-to-head comparison between the second-generation vector and a benchmark vector, identical in design to onasemnogene abeparvovec, the 2nd-generation vector showed better safety and improved efficacy in SMA mouse model.