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BACKGROUND: Immune checkpoint inhibitors are approved for the treatment of various tumors, but the response rate is not satisfactory in certain malignancies. Inhibitor of apoptosis proteins (IAP) ubiquitin-E3 ligase activity is involved in the regulation of immune responses. APG-1387 is a novel second mitochondria-derived activator of caspase (Smac) mimetic IAP inhibitor. The aim of this study was to explore the synergistic effect of APG-1387 when combined with anti-PD-1 antibody in a preclinical setting. METHODS: We utilized syngeneic mouse models of ovarian cancer (ID8), colon cancer (MC38), malignant melanoma (B16), and liver cancer (Hepa1-6) to assess the combination effect of APG-1387 and anti-PD-1 antibody, including immune-related factors, tumor growth, and survival. MSD V-PLEX validated assays were used to measure in vitro and in vivo cytokine release. RESULTS: In ID8 ovarian cancer and MC38 colon cancer models, APG-1387 and anti-PD1 antibody had synergistic antitumor effects. In the MC38 model, the combination of APG-1387 and anti-PD-1 antibody significantly inhibited tumor growth (P < 0.0001) and increased the survival rate of tumor-bearing animals (P < 0.001). Moreover, we found that APG-1387 upregulated tumor-infiltrating CD3 + NK1.1 + cells by nearly 2-fold, by promoting tumor cell secretion of IL-12. Blocking IL-12 secretion abrogated the synergistic effects of APG-1387 and anti-PD-1 antibody in both MC38 and ID8 models. CONCLUSIONS: APG-1387 has the potential to turn "cold tumors" into hot ones by recruiting more CD3 + NK1.1 + cells into certain tumors. Based on these and other data, the safety and therapeutic effect of this combination will be investigated in a phase 1/2 trial in patients with advanced solid tumors or hematologic malignancies (NCT03386526).
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Single-cell proteomics offers unparalleled insights into cellular diversity and molecular mechanisms, enabling a deeper understanding of complex biological processes at the individual cell level. Here, we develop an integrated sample processing on an active-matrix digital microfluidic chip for single-cell proteomics (AM-DMF-SCP). Employing the AM-DMF-SCP approach and data-independent acquisition (DIA), we identify an average of 2258 protein groups in single HeLa cells within 15 min of the liquid chromatography gradient. We performed comparative analyses of three tumor cell lines: HeLa, A549, and HepG2, and machine learning was utilized to identify the unique features of these cell lines. Applying the AM-DMF-SCP to characterize the proteomes of a third-generation EGFR inhibitor, ASK120067-resistant cells (67R) and their parental NCI-H1975 cells, we observed a potential correlation between elevated VIM expression and 67R resistance, which is consistent with the findings from bulk sample analyses. These results suggest that AM-DMF-SCP is an automated, robust, and sensitive platform for single-cell proteomics and demonstrate the potential for providing valuable insights into cellular mechanisms.
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We report a fully integrated digital microfluidic absorbance detection system with an enhanced sensitivity for online bacterial monitoring. Through a 100 µm gap in the chip, our optical detection system has a detection sensitivity for a BCA protein concentration of 0.1 mg mL-1. The absorbance detection limit of our system is 1.4 × 10-3 OD units, which is one order of magnitude better than that of the existing studies. The system's linear region is 0.1-7 mg mL-1, and the dynamic range is 0-25 mg mL-1. We measured the growth curves of wild-type and E. coli transformed with resistance plasmids and mixed at different ratios on chip. We sorted out the bacterial species including highly viable single cells based on the difference in absorbance data of growth curves. We explored the changes in the growth curves of E. coli under different concentrations of resistant media. In addition, we successfully screened for the optimal growth environment of the bacteria, in which the growth rate of PET30a-DH5α (in a medium with 33 µg mL-1 kanamycin resistance) was significantly higher than that of a 1 mg mL-1 resistance medium. In conclusion, the enhanced digital microfluidic absorbance detection system exhibits exceptional sensitivity, enabling precise bacterial monitoring and growth curve analysis, while also laying the foundation for DMF-based automated bioresearch platforms, thus advancing research in the life sciences.
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Microfluídica , Dispositivos Ópticos , Escherichia coli , Dispositivos Lab-On-A-Chip , Movimento CelularRESUMO
Background: Accumulating evidence has revealed that CD8+ T cell exhaustion (Tex) results in worse immunotherapy outcomes. However, the molecular functions and mechanisms of action of Tex in chemoresistance needed to be elucidated. Methods: The populations of tumor-infiltrating CD8+ T cells (TILCD8Ts) in chemoresistant and chemosensitive groups of the GSE25066 dataset were calculated using CIBERSORT. Differentially expressed genes (DEGs) between TILCD8Ts and other immune cells were explored by integrating 16 immune cell datasets downloaded from the gene expression omnibus (GEO) database. Gene ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, univariate and multivariate Cox regression, and least absolute shrinkage and selection operator (LASSO) regression of TILCD8T-specific upregulated genes were used to construct a chemoresistant TILCD8T signature (cr-TILCD8TSig). Clinical prognostic data, genomic alterations, chemotherapy response, and immunotherapy response were compared between the different cr-TILCD8TSig subgroups in the GSE25066 and the cancer genome atlas breast cancer (TCGA-BRCA) cohorts. Results: A cr-TILCD8TSig with exhausted features was identified, consisting of seven genes (TCF7, RARRES3, ARL4C, ITK, CDH3, GZMB, and KLRD1), which were identified from 104 TILCD8Ts-specific DEGs. Our results showed that compared to the cr-TILCD8TSig-low subgroup, the -high subgroup had a poorer distant relapse-free survival (DRFS) in the GSE25066 cohort and worse progression-free survival (PFS) in the TCGA-BRCA cohort. Univariate and multivariate Cox regression analyses also demonstrated that cr-TILCD8TSig was an independent prognostic factor in the two independent cohorts. Furthermore, cr-TILCD8TSig-low patients benefited more from chemotherapy and immunotherapy than cr-TILCD8TSig-high patients. Besides, we found cell transmembrane signal transduction and the ECM may provide the molecular basis for resistance to antitumor agents in the cr-TILCD8Sig-high subgroup. For genomic alterations, we revealed that mutations in PIK3CA, DMD, and APOB were more common in the cr-TILCD8Sig-high subgroup than in the cr-TILCD8Sig-low subgroup. A nomogram was finally constructed with good discrimination and calibration. Conclusions: cr-TILCD8TSig is a useful tool to independently predict prognosis, chemotherapy response, and immunotherapy outcomes in patients with breast cancer.
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Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Recidiva Local de Neoplasia , Linfócitos T CD8-Positivos , Calibragem , Fatores de Ribosilação do ADPRESUMO
BACKGROUND: Hepatitis B surface antigen (HBsAg) is a detectable index after hepatitis B virus (HBV) infection, which is a risk factor of hepatocellular carcinoma (HCC). However, few studies have focused on the expression of HBsAg in HCC patients' liver tissues. This study aimed to explore the potential utility of using HBsAg protein expression in normal liver tissues as a prognostic factor for HCC patients who underwent liver resection. STUDY DESIGN: The study enrolled 100 HCC patients with seropositivity for HBsAg. The liver tissues were collected, and tissue microarrays were constructed. The expression of HBsAg in liver tissues were measured by immunohistochemistry (IHC). Relevant clinical data and follow-up records were collected for analysis. RESULTS: HBsAg expressions was detected in 29 patients (positive group) and was unable to be detected in the remaining 71 patients (negative group). The patients in the positive group had higher HBV DNA levels (P < 0.05) than the patients in the negative group. The overall survival (OS) rate of the positive group was worse than the OS rate of the negative group (P = 0.013). The OS rates after resection at 1 and 2 years in negative group were 90.1% and 85.7%, respectively, while the value in the positive group were 79.3% and 65.5%, respectively. Multivariate analysis showed that HBsAg expression in liver tissues, ascites and alpha-fetoprotein levels were independent factors influencing OS. Similarly, after propensity score matching (PSM), the OS was worse in the positive group than in the negative group, and HBsAg expression could also serve as a predictor for OS (P = 0.039). The OS rates after resection and PSM at 1 and 2 years were 93.2% and 85.9% in the negative group, while the value in the positive group were 79.3% and 65.5%. CONCLUSION: As determined according to grouping based on immunohistochemistry staining results for HBsAg, this study indicated that HBsAg expression in liver tissues could predict the OS of HBV-related HCC patients after liver resection.
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Carcinoma Hepatocelular , Hepatite B Crônica , Hepatite B , Neoplasias Hepáticas , Carcinoma Hepatocelular/cirurgia , Hepatite B/complicações , Hepatite B/diagnóstico , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/cirurgia , PrognósticoRESUMO
Radiotherapy (RT) is a key component of neoadjuvant chemoradiotherapy to treat locally advanced rectal cancer (LARC). However, the therapeutic effect is limited due to radioresistance. Investigating the biomarkers of radioresistance might assist in the development of more effective therapeutic strategies for LARC.In this study, we investigated the different gene expressions in tumor samples from 110 patients using transcriptome analysis and immunohistochemistry (IHC), and identified serum- and glucocorticoid-regulated kinase 1 (SGK1) as a modulator of LARC radioresistance. We evaluated the impact of genetic and pharmacologic inhibition of the gene associated with radioresistance in vitro and in vivo. We found that the expression of SGK1 was upregulated in non-pathological complete response (non-pCR) patients. A high SGK1 expression was associated with radioresistance, whereas the genetic or pharmacologic inhibition of SGK1 expression reduced the radioresistance. We found that activate transcription factor 3 (ATF3) is a regulator of SGK1 in radioresistance.In conclusion, our findings indicate that SGK1 is a key player in LARC radioresistance, and drives radioresistance in an ATF3 dependent manner, which provides insights for future radio-sensitizer design.
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Regulação Neoplásica da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/radioterapia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas Imediatamente Precoces/genética , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Experimentais , Proteínas Serina-Treonina Quinases/genéticaRESUMO
It is well known that the aberrant expression of programmed death ligand 1 (PD-L1) on tumor cells impairs antitumor immunity. To date, in hepatocellular carcinoma (HCC), the relationship between PD-L1 expression and host-tumor immunity is not well defined. Here, the expression levels of PD-L1 and CD8(+) T cell infiltration were analyzed by immunohistochemistry (IHC) in formalin fixed paraffin embedded (FFPE) specimens from 167 HCC patients undergoing resection. A significant positive association was found between PD-L1 expression and the presence of CD8(+) T cell (p < 0.0001). Moreover, constitutive PD-L1 protein expression was not detected by western blot in HepG2, Hep3B, and 7402 HCC cancer cell lines; but co-cultured these cell lines with INFγ, a cytokine produced by activated CD8(+) T cells, remarkably upregulated PD-L1 expression. In fresh frozen HCC specimens, INFγ was found to be significantly correlated with PD-L1 and CD8(+) gene expression, as evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). These findings indicate that increased PD-L1 level may represent an adaptive immune resistance mechanism exerted by tumor cells in response to endogenous antitumor activity. Both increased intratumoral PD-L1 and CD8(+) were significantly associated with superior DFS (CD8(+): p = 0.03; PD-L1: p = 0.023) and OS (CD8(+): p = 0.001 and PD-L1: p = 0.059), but PD-L1 expression was not independently prognostic. In conclusions, PD-L1 upregulation is mainly induced by activated CD8(+) cytotoxic T cells pre-existing in HCC milieu rather than be constitutively expressed by the tumor cells, and it is a favorable prognostic factor for HCC.
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Currently, the best clinical predictor for inflammatory bowel disease (IBD) is family history. Over 163 sequence variants have been associated with IBD in genome-wide association studies, but they have weak effects and explain only a fraction of the observed heritability. It is expected that additional variants contribute to the genomic architecture of IBD, possibly including rare variants with effect sizes larger than the identified common variants. Here we applied a family study design and sequenced 38 individuals from five families, under the hypothesis that families with multiple IBD-affected individuals harbor one or more risk variants that (i) are shared among affected family members, (ii) are rare and (iii) have substantial effect on disease development. Our analysis revealed not only novel candidate risk variants but also high polygenic risk scores for common known risk variants in four out of the five families. Functional analysis of our top novel variant in the remaining family, a rare missense mutation in the ubiquitin ligase TRIM11, suggests that it leads to increased nuclear factor of kappa light chain enhancer in B-cells (NF-κB) signaling. We conclude that an accumulation of common weak-effect variants accounts for the high incidence of IBD in most, but not all families we analyzed and that a family study design can identify novel rare variants conferring risk for IBD with potentially large effect size, such as the TRIM11 p.H414Y mutation.
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UNLABELLED: Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. IMPORTANCE: Posttranslational modification of proteins enables cells to respond quickly to infections and immune stimuli in a tightly controlled manner. Specifically, covalent modification of proteins with the small protein ubiquitin is essential for cells to initiate and terminate immune signaling in response to bacterial and viral infection. This process is controlled by ubiquitin ligase enzymes, which themselves must be regulated to prevent persistent and deleterious immune signaling. However, how this regulation is achieved is poorly understood. This paper reports a novel ubiquitination event of the atypical ubiquitin ligase HOIP that is required to terminate bacterial lipopolysaccharide (LPS)-induced TLR4 immune signaling. Ubiquitination causes the HOIP ligase to undergo a conformational change, which blocks its enzymatic activity and ultimately terminates LPS-induced TLR4 signaling. These findings provide a new mechanism for controlling HOIP ligase activity that is vital to properly regulate a proinflammatory immune response.
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Multimerização Proteica , Processamento de Proteína Pós-Traducional , Receptor 4 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Imunidade Inata , Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , Conformação Proteica , Ubiquitina-Proteína Ligases/químicaRESUMO
UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298-5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE: The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.
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Herpesvirus Humano 8/fisiologia , Proteínas Oncogênicas/fisiologia , Proteínas Virais/fisiologia , Replicação Viral/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Apoptose , Autofagia , Sequência de Bases , Linhagem Celular , Replicação do DNA , DNA Viral/genética , Expressão Gênica , Técnicas de Inativação de Genes , Genoma Viral , Células HEK293 , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Replicação Viral/genéticaRESUMO
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.
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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
The MB21D1/cGAS (Mab-21 domain-containing 1/cyclic GMP-AMP [cGAMP] synthetase), acts as an intracellular pattern recognition receptor (PPR) to sense cytosolic pathogen DNAs and subsequently generates the second messenger cGAMP to initiate the TMEM173/STING pathway for interferon (IFN) production. Intriguingly, we have recently demonstrated crosstalk between the intracellular DNA sensing pathway and autophagy machinery by demonstrating a direct interaction between the MB21D1 DNA sensor and the BECN1/Beclin 1 autophagy protein. This interaction not only suppresses MB21D1 enzymatic activity to halt cGAMP production, but also enhances the autophagy-mediated degradation of cytosolic microbial DNAs. This demonstrates that MB21D1 is the molecular link between the intracellular DNA sensing pathway and the autophagy pathway, ultimately developing well-balanced immune responses against pathogens.
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Proteínas Reguladoras de Apoptose/imunologia , DNA/imunologia , Herpesvirus Humano 1/imunologia , Imunidade Inata , Proteínas de Membrana/imunologia , Nucleotidiltransferases/imunologia , Animais , HumanosRESUMO
Linear ubiquitination is a newly discovered posttranslational modification that is currently restricted to a small number of known protein substrates. The linear ubiquitination assembly complex (LUBAC), consisting of HOIL-1L, HOIP, and Sharpin, has been reported to activate NF-κB-mediated transcription in response to receptor signaling by ligating linear ubiquitin chains to Nemo and Rip1. Despite recent advances, the detailed roles of LUBAC in immune cells remain elusive. We demonstrate a novel HOIL-1L function as an essential regulator of the activation of the NLRP3/ASC inflammasome in primary bone marrow-derived macrophages (BMDMs) independently of NF-κB activation. Mechanistically, HOIL-1L is required for assembly of the NLRP3/ASC inflammasome and the linear ubiquitination of ASC, which we identify as a novel LUBAC substrate. Consequently, we find that HOIL-1L(-/-) mice have reduced IL-1ß secretion in response to in vivo NLRP3 stimulation and survive lethal challenge with LPS. Together, these data demonstrate that linear ubiquitination is required for NLRP3 inflammasome activation, defining the molecular events of NLRP3 inflammasome activation and expanding the role of LUBAC as an innate immune regulator. Furthermore, our observation is clinically relevant because patients lacking HOIL-1L expression suffer from pyogenic bacterial immunodeficiency, providing a potential new therapeutic target for enhancing inflammation in immunodeficient patients.
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Proteínas de Transporte/imunologia , Imunidade Inata/fisiologia , Inflamassomos/imunologia , Macrófagos/imunologia , Complexos Multiproteicos/imunologia , Ubiquitinação/fisiologia , Animais , Proteínas de Transporte/genética , Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/imunologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamassomos/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lipopolissacarídeos/toxicidade , Macrófagos/citologia , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , NF-kappa B/genética , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologiaRESUMO
Robust immune responses are essential for eliminating pathogens but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) produces the second messenger cGAMP to initiate the stimulator of interferon genes (STING) pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double-stranded DNA (dsDNA) stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNA to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNA. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced antimicrobial immune responses.
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Proteínas Reguladoras de Apoptose/imunologia , DNA/imunologia , Herpesvirus Humano 1/imunologia , Imunidade Inata , Proteínas de Membrana/imunologia , Nucleotidiltransferases/imunologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia , Proteína Beclina-1 , Linhagem Celular , DNA/metabolismo , Humanos , Interferons/imunologia , Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/metabolismo , Fosfatidilinositol 3-Quinase/metabolismoRESUMO
Autophagy is an important innate safeguard mechanism for protecting an organism against invasion by pathogens. We have previously discovered that Kaposi's sarcoma-associated herpesvirus (KSHV) evades this host defense through tight suppression of autophagy by targeting multiple steps of autophagy signal transduction. Here, we report that KSHV K7 protein interacts with Rubicon autophagy protein and inhibits the autophagosome maturation step by blocking Vps34 enzymatic activity, further highlighting how KSHV deregulates autophagy-mediated host immunity for its life cycle.
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Autofagia , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Fagossomos/metabolismo , Sarcoma de Kaposi/patologia , Proteínas Virais/metabolismo , Proteínas Relacionadas à Autofagia , Células HeLa , Humanos , Immunoblotting , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Transdução de SinaisRESUMO
Vesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) regulate intracellular cholesterol homeostasis, which is required for many virus infections. During entry, viruses or virus-containing vesicles can fuse with endosomal membranes to mediate the cytosolic release of virions, and alterations in endosomal cholesterol can inhibit this invasion step. We show that the antiviral effector protein interferon-inducible transmembrane protein 3 (IFITM3) interacts with VAPA and prevents its association with OSBP, thereby disrupting intracellular cholesterol homeostasis and inhibiting viral entry. By altering VAPA-OSBP function, IFITM3 induces a marked accumulation of cholesterol in multivesicular bodies and late endosomes, which inhibits the fusion of intraluminal virion-containing vesicles with endosomal membranes and thereby blocks virus release into the cytosol. Consequently, ectopic expression or depletion of the VAPA gene profoundly affects IFITM3-mediated inhibition of viral entry. Thus, IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry, further underscoring the importance of cholesterol in virus infection.
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Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Vírion/fisiologia , Internalização do Vírus , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Citosol/virologia , Endossomos/metabolismo , Endossomos/virologia , Células HEK293 , Homeostase/fisiologia , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Proteínas R-SNARE/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesiculovirus/metabolismo , Vesiculovirus/fisiologia , Vírion/metabolismoRESUMO
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.
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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
Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway.
Assuntos
Interferon Tipo I/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Humanos , Camundongos , Mutação , Receptores do Ácido Retinoico/genética , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genéticaRESUMO
Induction of Type I IFNs is a central event in antiviral responses and must be tightly controlled. The protein kinase TBK1 is critically involved in virus-triggered type I IFN signaling. In this study, we identify an alternatively spliced isoform of TBK1, termed TBK1s, which lacks exons 3-6. Upon Sendai virus (SeV) infection, TBK1s is induced in both human and mouse cells and binds to RIG-1, disrupting the interaction between RIG-I and VISA. Consistent with that result, overexpression of TBK1s inhibits IRF3 nuclear translocation and leads to a shutdown of SeV-triggered IFN-beta production. Taken together, our data indicate that TBK1s plays an inhibitory role in virus-triggered IFN-beta signaling pathways.
Assuntos
Processamento Alternativo , Interferon beta/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Infecções por Respirovirus/metabolismo , Vírus Sendai , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Indução Enzimática , Humanos , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Receptores de Superfície Celular , Receptores do Ácido Retinoico/metabolismoRESUMO
Toll-like receptor (TLR) signaling is pivotal to innate and adaptive immune responses and must be tightly controlled. The mechanisms of TLR signaling have been the focus of extensive studies. Here we report that the tripartite-motif protein TRIM30alpha, a RING protein, was induced by TLR agonists and interacted with the TAB2-TAB3-TAK1 adaptor-kinase complex involved in the activation of transcription factor NF-kappaB. TRIM30alpha promoted the degradation of TAB2 and TAB3 and inhibited NF-kappaB activation induced by TLR signaling. In vivo studies showed that transfected or transgenic mice overexpressing TRIM30alpha were more resistant to endotoxic shock. Consistent with that, in vivo 'knockdown' of TRIM30alpha mRNA by small interfering RNA impaired lipopolysaccharide-induced tolerance. Finally, expression of TRIM30alpha depended on NF-kappaB activation. Our results collectively indicate that TRIM30alpha negatively regulates TLR-mediated NF-kappaB activation by targeting degradation of TAB2 and TAB3 by a 'feedback' mechanism.