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Hepatocellular carcinoma (HCC) still presents poor prognosis with low overall survival rates and limited therapeutic options available. Recently, attention has been drawn to peptidomic analysis, an emerging field of proteomics for the exploration of new potential peptide drugs for the treatment of various diseases. However, research on the potential function of HCC peptides is lacking. Here, we analyzed the peptide spectrum in HCC tissues using peptidomic techniques and explored the potentially beneficial peptides involved in HCC. Changes in peptide profiles in HCC were examined using liquid chromatography-mass spectrometry (LC-MS/MS). Analyze the physicochemical properties and function of differently expressed peptides using bioinformatics. The effect of candidate functional peptides on HCC cell growth and migration was evaluated using the CCK-8, colony formation, and transwell assays. Transcriptome sequencing analysis and western blot were employed to delve into the mode of action of potential peptide on HCC. Peptidomic analysis of HCC tissue yielded a total of 8683 peptides, of which 452 exhibited up-regulation and 362 showed down-regulation. The peptides that were differentially expressed, according to bioinformatic analysis, were closely linked to carbon metabolism and the mitochondrial inner membrane. The peptide functional validation identified a novel peptide, PDLC (peptide derived from liver cancer), which was found to dramatically boost HCC cell proliferation through the Ras/Raf/MEK/ERK signaling cascade. Our research defined the peptide's properties and pattern of expression in HCC and identified a novel peptide, PDLC, with a function in encouraging HCC progression, offering an entirely new potential therapeutic target the disease.
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Carcinoma Hepatocelular , Proliferação de Células , Neoplasias Hepáticas , Sistema de Sinalização das MAP Quinases , Proteômica , Quinases raf , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Proliferação de Células/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases raf/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Proteínas ras/metabolismo , Proteínas ras/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Movimento Celular/efeitos dos fármacos , Espectrometria de Massas em Tandem , Regulação Neoplásica da Expressão GênicaRESUMO
Myeloid-derived suppressor cells (MDSCs) occupy a pivotal role in the intricate pathogenesis of the autoimmune disorder, Type 1 diabetes mellitus (T1DM). Since our previous work demonstrated that trichosanthin (TCS), an active compound of Chinese herb medicine Tian Hua Fen, regulated immune response, we aimed to clarify the efficacy and molecular mechanism of TCS in the treatment of T1DM. To this end, T1DM mouse model was established by streptozotocin (STZ) induction. The mice were randomly divided into normal control group (Ctl), T1DM group (STZ), TCS treated diabetic group (STZ + TCS) and insulin-treated diabetic group (STZ + insulin). Our comprehensive evaluation encompassed variables such as blood glucose, glycosylated hemoglobin, body weight, pertinent biochemical markers, pancreatic histopathology, and the distribution of immune cell populations. Furthermore, we meticulously isolated MDSCs from the bone marrow of T1DM mice, probing into the expressions of genes pertaining to the advanced glycation end product receptor (RAGE)/NF-κB signaling pathway through RT-qPCR. Evidently, TCS exhibited a substantial capacity to effectively counteract the T1DM-induced elevation in random blood glucose, glycosylated hemoglobin, and IL-6 levels in plasma. Pathological scrutiny underscored the ability of TCS to mitigate the damage incurred by islets. Intriguingly, TCS interventions engendered a reduction in the proportion of MDSCs within the bone marrow, particularly within the IL-6+ MDSC subset. In contrast, IL-10+ MDSCs exhibited an elevation following TCS treatment. Moreover, we observed a significant down-regulation of relative mRNA of pro-inflammatory genes, including arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), RAGE and NF-κB, within MDSCs due to the influence of TCS. It decreases total MDSCs and regulates the balance between IL-6+ and IL-10+ MDSCs thus alleviating the symptoms of T1DM. TCS also down-regulates the RAGE/NF-κB signaling pathway, making it a promising alternative therapeutic treatment for T1DM. Collectively, our study offered novel insights into the underlying mechanism by which TCS serves as a promising therapeutic intervention for T1DM.
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Uncontrolled and excessive progression of liver fibrosis is thought to be the prevalent pathophysiological cause of liver cirrhosis and hepatocellular cancer, and there are currently no effective antifibrotic therapeutic options available. Intercellular communication and cellular heterogeneity in the liver are involved in the progression of liver fibrosis, but the exact nature of the cellular phenotypic changes and patterns of interregulatory remain unclear. Here, we performed single-cell RNA sequencing on nonparenchymal cells (NPCs) isolated from normal and fibrotic mouse livers. We identified eight main types of cells, including endothelial cells, hepatocytes, dendritic cells, B cells, natural killer/T (NK/T) cells, hepatic stellate cells (HSCs), cholangiocytes and macrophages, and revealed that macrophages and HSCs exhibit the most variance in transcriptional profile. Further analyses of HSCs and macrophage subpopulations and ligand-receptor interaction revealed a high heterogeneity characterization and tightly interregulated network of these two groups of cells in liver fibrosis. Finally, we uncovered a profibrotic Thbs1+ macrophage subcluster, which expands in mouse and human fibrotic livers, activating HSCs via PI3K/AKT/mTOR signaling pathway. Our findings decode unanticipated insights into the heterogeneity of HSCs and macrophages and their intercellular crosstalk at a single-cell level, and may provide potential therapeutic strategies in liver fibrosis.
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Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and is associated with high mortality. Transmembrane protein 147 (TMEM147) is a seven-transmembrane protein that may mediate immune regulation. However, the relevance of TMEM147 to immune regulation in HCC and the prognosis of HCC patients are unclear. Methods: We analyzed TMEM147 expression in HCC by using the Wilcoxon rank-sum test. Real time quantitative PCR (RT-qPCR) and Western blot analysis of tumor tissues and cell lines were used to verify TMEM147 expression in HCC. The influence of TMEM147 on HCC prognosis was assessed using Kaplan-Meier analysis, Cox regression analysis, and a prognostic nomogram. The functions of the TMEM147-related differentially expressed genes (DEGs) were identified by Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and gene set enrichment analysis (GSEA). In addition, we examined the associations between TMEM147 expression and immune infiltration using single-sample gene set enrichment analysis (ssGSEA) and immunofluorescence staining of HCC tissues. Results: Our results showed that the expression of TMEM147 was significantly higher in human HCC tissues than in adjacent normal liver tissues, with similar findings in human HCC cell lines. High TMEM147 expression was correlated with T stage, pathological stage, histological grade, race, alpha-fetoprotein level, and vascular invasion in HCC. Moreover, we revealed that high TMEM147 expression was associated with shorter survival times and that TMEM147 could be a risk factor for overall survival, along with T stage, M stage, pathological stage, and tumor status. Mechanistic studies revealed that high TMEM147 expression was linked to the B lymphocyte, antigen response, IL6 signaling pathway, cell cycle, Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling pathway, and myelocytomatosis oncogene (MYC) targets. Correspondingly, TMEM147 expression was positively associated with the infiltration of immune cells, including Th2 cells, follicular helper T cells, macrophages, and NK CD56 bright cells in HCC. Conclusions: TMEM147 might be a biomarker for poor prognosis and is related to immune cell infiltration in HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Linfócitos B , Western Blotting , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , PrognósticoRESUMO
Although B lymphocytes are widely known to participate in the immune response, the conclusive roles of B lymphocyte subsets in the antitumor immune response have not yet been determined. Single-cell data from GEO datasets were first analyzed, and then a B cell flow cytometry panel was used to analyze the peripheral blood of 89 HCC patients and 33 healthy controls recruited to participate in our research. Patients with HCC had a higher frequency of B10 cells and a lower percentage of MZB cells than healthy controls. And the changes in B cell subsets might occur at an early stage. Moreover, the frequency of B10 cells decreased after surgery. Positively correlated with B10 cells, the elevated IL-10 level in HCC serum may be a new biomarker in HCC identification. For the first time, our results suggest that altered B cell subsets are associated with the development and prognosis of HCC. Increased B10 cell percentage and IL-10 in HCC patients suggest they might augment the development of liver tumors. Hence, B cell subsets and related cytokines may have predictive value in HCC patients and could be potential targets for immunotherapy in HCC.
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Subpopulações de Linfócitos B , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Interleucina-10 , CitocinasRESUMO
Background and Aims: The Quzhi formula, a Chinese medicine compound prescription, relieves nonalcoholic steatohepatitis (NASH) symptoms. This study aimed to explore the mechanism of the Quzhi formula against NASH. Methods: A choline-deficient, L-amino acid-defined, high-fat diet induced a NASH mouse model and a free fatty acid-induced mouse hepatocyte cell model were used to evaluate the function of Quzhi formula in vivo and in vitro. Network pharmacology and molecular docking technology were performed to uncover the possible protective mechanisms of the Quzhi formula against NASH. Key factors in liver lipid metabolism and endoplasmic reticulum (ER) stress pathway were evaluated to verify the mechanism. Results: The positive contribution of the Quzhi formula on NASH was confirmed in vivo and in vitro. Abnormal accumulation of lipid in the liver and inflammatory responses were significantly decreased by the Quzhi formula. Network pharmacological analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the Quzhi formula protected against NASH by regulating ER stress and inflammatory responses, which was enhanced by further molecular docking analysis. In addition, mechanism exploration showed that Quzhi formula mainly reduced ER stress by downregulating Bip/eIF2α signaling. Conclusions: The Quzhi formula protected against NASH by inhibiting lipid accumulation, ER stress, and inflammatory responses, which supports the potential use of Quzhi formula as an alternative treatment for NASH.
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Acute lung injury (ALI) is a severe clinical disease marked by uncontrolled inflammation response which lacks effective medicines. Accumulative evidence has indicated that macrophages are therapeutic targets for treating ALI because of its critical role in the inflammatory response.Palmatine (PAL), an isoquinoline alkaloid extracted from natural plants, exhibits effective anti-inflammatory, anti-tumor, and anti-oxidation activities. Here we reported that PAL alleviated LPS-induced acute lung injury and attenuated inflammatory cell infiltration especially neutrophils. Moreover, PAL also attenuated the production of TNF-α, CXCL-1, CXCL-2 and nitric oxide in bronchoalveolar lavage fluid. In addition, PAL remarkably reduced LPS-induced expression of TNF-α, CXCL-1 and CXCL-2 in bone marrow derived macrophages (BMDMs) and alveolar macrophages (AMs). Treatment with PAL inhibited the phosphorylation and interaction of TAK1/TAB1, which in turn attenuated the p38 MAPK and NF-κB signal pathways in BMDMs. Our results indicated that PAL ameliorated LPS-induced ALI by inhibiting macrophage activation through inhibiting NF-κB and p38 MAPK pathways, suggesting that PAL has anti-inflammation effect on ALI.
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Lesão Pulmonar Aguda , Lipopolissacarídeos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alcaloides de Berberina , Citocinas/metabolismo , Humanos , Pulmão , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (named COVID-ONE-hi). COVID-ONE-hi is based on the data that contain the IgG/IgM responses to 24 full-length/truncated proteins corresponding to 20 of 28 known SARS-CoV-2 proteins and 199 spike protein peptides against 2360 serum samples collected from 783 COVID-19 patients. In addition, 96 clinical parameters for the 2360 serum samples and basic information for the 783 patients are integrated into the database. Furthermore, COVID-ONE-hi provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the "START" button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-hi is freely available at www.COVID-ONE.cn.
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COVID-19 , Anticorpos Antivirais , Humanos , Imunidade Humoral , Imunoglobulina G , Imunoglobulina M , SARS-CoV-2RESUMO
The immunogenicity of the SARS-CoV-2 proteome is largely unknown, especially for non-structural proteins and accessory proteins. In this study, we collect 2,360 COVID-19 sera and 601 control sera. We analyze these sera on a protein microarray with 20 proteins of SARS-CoV-2, building an antibody response landscape for immunoglobulin (Ig)G and IgM. Non-structural proteins and accessory proteins NSP1, NSP7, NSP8, RdRp, ORF3b, and ORF9b elicit prevalent IgG responses. The IgG patterns and dynamics of non-structural/accessory proteins are different from those of the S and N proteins. The IgG responses against these six proteins are associated with disease severity and clinical outcome, and they decline sharply about 20 days after symptom onset. In non-survivors, a sharp decrease of IgG antibodies against S1 and N proteins before death is observed. The global antibody responses to non-structural/accessory proteins revealed here may facilitate a deeper understanding of SARS-CoV-2 immunology.
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COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas não Estruturais Virais/imunologia , Proteínas Virais Reguladoras e Acessórias/imunologia , Adulto , Idoso , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Análise Serial de ProteínasRESUMO
To fully decipher the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein, it is essential to assess which part is highly immunogenic in a systematic way. We generate a linear epitope landscape of the Spike protein by analyzing the serum immunoglobulin G (IgG) response of 1,051 coronavirus disease 2019 (COVID-19) patients with a peptide microarray. We reveal two regions rich in linear epitopes, i.e., C-terminal domain (CTD) and a region close to the S2' cleavage site and fusion peptide. Unexpectedly, we find that the receptor binding domain (RBD) lacks linear epitope. We reveal that the number of responsive peptides is highly variable among patients and correlates with disease severity. Some peptides are moderately associated with severity and clinical outcome. By immunizing mice, we obtain linear-epitope-specific antibodies; however, no significant neutralizing activity against the authentic virus is observed for these antibodies. This landscape will facilitate our understanding of SARS-CoV-2-specific humoral responses and might be useful for vaccine refinement.
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COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , COVID-19/epidemiologia , COVID-19/genética , China/epidemiologia , Modelos Animais de Doenças , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Genetic heterogeneity of tumor is closely related to its clonal evolution, phenotypic diversity and treatment resistance, and such heterogeneity has only been characterized at single-cell sub-chromosomal scale in liver cancer. Here we reconstructed the single-variant resolution clonal evolution in human liver cancer based on single-cell mutational profiles. The results indicated that key genetic events occurred early during tumorigenesis, and an early metastasis followed by independent evolution was observed in primary liver tumor and intrahepatic metastatic portal vein tumor thrombus. By parallel single-cell RNA-Seq, the transcriptomic phenotype of HCC was found to be related with genetic heterogeneity. For the first time we reconstructed the single-cell and single-variant clonal evolution in human liver cancer, and dissection of both genetic and phenotypic heterogeneity will facilitate better understanding of their relationship.
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Carcinoma Hepatocelular/genética , Evolução Clonal , Neoplasias Hepáticas/genética , Humanos , Mutação , Análise de Célula Única , Células Tumorais CultivadasRESUMO
The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.
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Apolipoproteínas B/metabolismo , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Proteínas de Membrana/genética , Triglicerídeos/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Lipoproteínas VLDL/sangue , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/etiologia , Obesidade/metabolismo , Triglicerídeos/sangue , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Serological tests play an essential role in monitoring and combating the COVID-19 pandemic. Recombinant spike protein (S protein), especially the S1 protein, is one of the major reagents used for serological tests. However, the high cost of S protein production and possible cross-reactivity with other human coronaviruses pose unavoidable challenges. By taking advantage of a peptide microarray with full spike protein coverage, we analyzed 2,434 sera from 858 COVID-19 patients, 63 asymptomatic patients and 610 controls collected from multiple clinical centers. Based on the results, we identified several S protein-derived 12-mer peptides that have high diagnostic performance. In particular, for monitoring the IgG response, one peptide (aa 1148-1159 or S2-78) exhibited a sensitivity (95.5%, 95% CI 93.7-96.9%) and specificity (96.7%, 95% CI 94.8-98.0%) comparable to those of the S1 protein for the detection of both symptomatic and asymptomatic COVID-19 cases. Furthermore, the diagnostic performance of the S2-78 (aa 1148-1159) IgG was successfully validated by ELISA in an independent sample cohort. A panel of four peptides, S1-93 (aa 553-564), S1-97 (aa 577-588), S1-101 (aa 601-612) and S1-105 (aa 625-636), that likely will avoid potential cross-reactivity with sera from patients infected by other coronaviruses was constructed. The peptides identified in this study may be applied independently or in combination with the S1 protein for accurate, affordable, and accessible COVID-19 diagnosis.
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Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19 , COVID-19/sangue , Imunoglobulina G/sangue , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
BACKGROUND: Although significant progress has been made in understanding the mechanisms of steatosis and insulin resistance, the physiological functions of the epigenetic regulators in these processes remain largely elusive. METHODS: Hepatocyte-specific Arid1a knockout mice were administrated with high-fat diet (HFD) for 12â¯weeks, then insulin sensitivity was assessed by glucose tolerance test (GTT) and insulin tolerance test (ITT). The metabolism-related indicators were determined by employing a variety of biological methods, including histology, real-time PCR, enzyme-linked immunosorbent assay (ELISA), Western blotting assay, Chromatin immunoprecipitation (ChIP), RNA-seq and assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). FINDINGS: Hepatocyte-specific Arid1a deletion significantly increases susceptibility to develop hepatic steatosis, insulin resistance and inflammation in mice fed a HFD. In vitro, Arid1a deletion in isolated hepatocytes directly leads to free fatty acid-induced lipid accumulation and insulin resistance. Mechanically, Arid1a deficiency impairs fatty acid oxidation by downregulating PPARα and altering the epigenetic landscape of some metabolism genes. INTERPRETATION: These findings reveal that targeting Arid1a might be a promising therapeutic strategy for liver steatosis and insulin resistance. FUND: This work was supported by National Natural Science Foundation of China (81672772 and 81472621), China National Science and Technology Major Project for Prevention and Treatment of Infectious Diseases (No.2017ZX 10203207) and National Program on Key Research Project of China (grant no. 2016YFC0902701).
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Proteínas de Ligação a DNA/genética , Resistência à Insulina/genética , Metabolismo dos Lipídeos/genética , Proteínas Nucleares/genética , Animais , Suscetibilidade a Doenças , Glucose/metabolismo , Hepatócitos/metabolismo , Histonas/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores Ativados por Proliferador de Peroxissomo , Transdução de Sinais , Fatores de TranscriçãoRESUMO
BACKGROUND: A major facilitator superfamily transporter Dehp2 was recently shown to be playing an important role in transport and biodegradation of haloacids in Paraburkholderia caribensis MBA4, and Dehp2 is phylogenetically conserved in Burkholderia sensu lato. RESULTS: We designed both Burkholderia sensu stricto-specific and Paraburkholderia-specific qPCR assays based on dehp2 and 16S rRNA, and validated the qPCR assays in 12 bacterial strains. The qPCR assays could detect single species of Burkholderia sensu stricto or Paraburkholderia with high sensitivity and discriminate them in mixtures with high specificity over a wide dynamic range of relative concentrations. At relatively lower cost compared with sequencing-based approach, the qPCR assays will facilitate discrimination of Burkholderia sensu stricto and Paraburkholderia in a large number of samples. CONCLUSIONS: For the first time, we report the utilization of a haloacids transporter gene for discriminative purpose in Burkholderia sensu lato. This enables not only quick decision on proper handling of putative pathogenic samples in Burkholderia sensu stricto group but also future exploitation of relevant species in Paraburkholderia group for haloacids biodegradation purposes.
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Burkholderia/genética , Burkholderiaceae/genética , Proteínas de Transporte/genética , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Filogenia , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Ring finger protein 8 (RNF8), an FHA/RING domain containing E3 ubiquitin ligase, is critical in supporting genome integrity by facilitating the assembly of multiple DNA repair proteins at DNA lesions following DNA damage. In the present study, a search for novel binding partners of RNF8 was performed using a yeast twohybrid screening assay, and small ubiquitinlike modifier (SUMO)2/3 was identified as one of the major RNF8binding candidates. GST pulldown and immunoprecipitation assays revealed that RNF8 bound directly and noncovalently to SUMO2/3, but not to SUMO1, and that the FHA domain of RNF8 was required for the binding to SUMO2/3. Furthermore, RNF8 colocalized with SUMO2/3 at sites of DNA lesions in response to ionizing radiation, as revealed by immunofluorescence assay. Survival assay indicated that the depletion of RNF8 and SUMO2/3 resulted in decreased cellular resistance to genotoxic stress. These data suggested that the binding of RNF8 to SUMO2/3 promoted the response to DNA damage.
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Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Sobrevivência Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Mutagênicos/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Ubiquitina-Proteína Ligases , Ubiquitinas/genéticaRESUMO
Innate immunity provides immediate defense against viral infection. Influenza A virus (IAV) is able to get past the first line of defense. Elucidation of the molecular interaction between influenza factors and the newly recognized host players in the innate response might help in our understanding of the root causes of virulence and pathogenicity of IAV. In this study, we show that expression of miR-26a leads to a significant inhibition of IAV replication. miR-26a does not directly target IAV genome. Instead, miR-26a activates the type I interferon (IFN) signaling pathway and promotes the production of IFN-stimulated genes, thus suppressing viral replication. Furthermore, ubiquitin-specific protease 3 (USP3), a negative regulator of type I IFN pathway, is targeted by miR-26a upon IAV challenge. However, miR-26a is significantly downregulated during IAV infection. Thus, downregulation of miR-26a is a new strategy evolved by IAV to counteract cellular antiviral responses. Our findings indicate that delivery of miR-26a may be a potential strategy for anti-IAV therapies.
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Vírus da Influenza A/patogenicidade , Interferon-alfa/metabolismo , Interferon beta/metabolismo , MicroRNAs/metabolismo , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Interferon Tipo I/metabolismo , MicroRNAs/genética , Plasmídeos/genética , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Persistent or excess activation of NF-κB leads to cancer, autoimmune and inflammatory diseases. Therefore, activated NF-κB needs to be terminated after induction, which highlights the physiological significance of NF-κB-negative regulators. However, the molecular mechanisms that negatively regulate NF-κB are not well understood. Here, we report that Ring Finger Protein 8 (RNF8), an E3 ubiquitin ligase, inhibits TNFα-mediated NF-κB activation by targeting IκB kinase (IKK). Upon TNFα stimulation, RNF8 binds to the catalytic subunits of IKK complex, resulting in inhibition of IKKα/ß phosphorylation and subsequent NF-κB activation. RNF8 targets the IKK complex in a manner independent of its RING domain. We further provide evidence that the silencing of RNF8 results in enhanced TNFα-induced IKK activation, and an increase expression of NF-κB-induced inflammatory cytokine IL-8. Our study identifies a previously unrecognized role for RNF8 in the negative regulation of NF-κB activation by targeting and deactivating the IKK complex.
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Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND/AIMS: MicroRNA (miRNA) is a small non-coding RNA molecule that functions in regulation of gene expression by targeting mRNA to affect its stability and/or translation. The aim of this study was to evaluate the miRNAs involvement in gestational diabetes mellitus (GDM), a well known risk factor for fetal overgrowth. METHODS: Differential microRNA expression in placental tissues of normal controls and women with GDM were identified by miRNA micorarray analysis and further confirmed by quantitative real-time PCR (qRT-PCR) on an independent set of normal and GDM placental tissues. Target genes of microRNAs were bioinformatically predicted and verified in vitro by Western blotting. RESULTS: Our results uncovered 9 miRNAs that were significantly deregulated in GDM samples: miR-508-3p was up-regulated and miR-27a, miR-9, miR-137, miR-92a, miR-33a, miR-30d, miR-362-5p and miR-502-5p were down-regulated. Bioinformatic approaches revealed that the microRNAs signature identifies gene targets involved in EGFR (epidermal growth factor receptor)-PI3K (phosphoinositide 3-Kinase)-Akt (also known as protein kinase B) pathway, a signal cascade which plays important roles in placental development and fetal growth. We found that the protein levels of EGFR, PI3K and phospho-Akt were up-regulated and PIKfyve (a FYVE finger-containing phosphoinositide kinase), a negative regulator of EGFR signaling, was down-regulated significantly in GDM tissues. We also confirmed PIKfyve was a direct target of miR-508-3p. CONCLUSION: Our data identified a miRNA signature involvement in GDM which may contribute to macrosomia through enhancing EGFR signaling.
Assuntos
Diabetes Gestacional/genética , Macrossomia Fetal/etiologia , Macrossomia Fetal/genética , MicroRNAs/genética , Adulto , Estudos de Casos e Controles , Regulação para Baixo/genética , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Placenta , Gravidez , Proteínas Proto-Oncogênicas c-akt , Risco , Transdução de Sinais/genética , Regulação para Cima/genética , Adulto JovemRESUMO
TANK-binding kinase 1 (TBK1)-mediated induction of type I interferon (IFN) plays a critical role in host antiviral responses and immune homeostasis. The negative regulation of TBK1 activity is largely unknown. We report that suppressor of cytokine signaling 3 (SOCS3) inhibits the IFN-ß signaling pathway by promoting proteasomal degradation of TBK1. Overexpression and knockdown experiments indicated that SOCS3 is a negative regulator of IFN regulatory factor 3 (IRF3) phosphorylation and IFN-ß transcription. Moreover, SOCS3 directly associates with TBK1, and they colocalize in the cytoplasm. SOCS3 catalyzes K48-linked polyubiquitination of TBK1 at Lys341 and Lys344 and promotes subsequent TBK1 degradation. On the contrary, SOCS3 knockdown markedly increases the abundance of TBK1. Interestingly, both the BOX domain of SOCS3 and Ser172 phosphorylation of TBK1 are indispensable for the processes of ubiquitination and degradation. Ectopic expression of SOCS3 significantly inhibits vesicular stomatitis virus (VSV) and influenza A virus strain A/WSN/33 (WSN)-induced IRF3 phosphorylation and facilitates the replication of WSN virus by detecting the transcription of its viral RNA (vRNA). Knockdown of SOCS3 represses WSN replication. Collectively, these results demonstrate that SOCS3 acts as a negative regulator of IFN-ß signal by ubiquitinating and degrading TBK1, shed light on the understanding of antiviral innate immunity, and provide a potential target for developing antiviral agents.