Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.
Humans ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms/pathology* ; Lysine/metabolism* ; NF-kappa B/metabolism* ; TNF Receptor-Associated Factor 6/metabolism* ; Transcription Factor AP-1/metabolism* ; Ubiquitin/metabolism*

The study investigated the inhibitory effect and mechanism of tectorigenin derivative(SGY) against herpes simplex virus type Ⅰ(HSV-1) by in vitro experiments. The cytotoxicity of SGY and positive drug acyclovir(ACV) on African green monkey kidney(Vero) cells and mouse microglia(BV-2) cells was detected by cell counting kit-8(CCK-8) method, and the maximum non-toxic concentration and median toxic concentration(TC_(50)) of the drugs were calculated. After Vero cells were infected with HSV-1, the virulence was determined by cytopathologic effects(CPE) to calculate viral titers. The inhibitory effect of the tested drugs on HSV-1-induced cytopathy in Vero cells was measured, and their modes of action were initially explored by virus adsorption, replication and inactivation. The effects of the drugs on viral load of BV-2 cells 24 h after HSV-1 infection and the Toll-like receptor(TLR) mRNA expression were detected by real-time fluorescence quantitative PCR(RT-qPCR). The maximum non-toxic concentrations of SGY against Vero and BV-2 cells were 382.804 μg·mL~(-1) and 251.78 μg·mL~(-1), respectively, and TC_(50) was 1 749.98 μg·mL~(-1) and 2 977.50 μg·mL~(-1), respectively. In Vero cell model, the half maximal inhibitory concentration(IC_(50)) of SGY against HSV-1 was 54.49 μg·mL~(-1), and the selection index(SI) was 32.12, with the mode of action of significantly inhibiting replication and directly inactivating HSV-1. RT-qPCR results showed that SGY markedly reduced the viral load in cells. The virus model group had significantly increased relative expression of TLR2, TLR3 and tumor necrosis factor receptor-associated factor 3(TRAF3) and reduced relative expression of TLR9 as compared with normal group, and after SGY intervention, the expression of TLR2, TLR3 and TRAF3 was decreased to different degrees and that of TLR9 was enhanced. The expression of inflammatory factors inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) was remarkably increased in virus model group as compared with that in normal group, and the levels of these inflammatory factors dropped after SGY intervention. In conclusion, SGY significantly inhibited and directly inactivated HSV-1 in vitro. In addition, it modulated the expression of TLR2, TLR3 and TLR9 related pathways, and suppressed the increase of inflammatory factor levels.
Animals ; Antiviral Agents/therapeutic use* ; Chlorocebus aethiops ; Herpes Simplex/pathology* ; Herpesvirus 1, Human/metabolism* ; Isoflavones ; Mice ; TNF Receptor-Associated Factor 3/pharmacology* ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 3/metabolism* ; Toll-Like Receptor 9/metabolism* ; Toll-Like Receptors/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Vero Cells ; Virus Replication

Tripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.
A549 Cells ; Animals ; Apoptosis Regulatory Proteins/immunology* ; DEAD Box Protein 58/immunology* ; Dogs ; HEK293 Cells ; Humans ; Influenza A Virus, H1N1 Subtype/immunology* ; Madin Darby Canine Kidney Cells ; Mice ; Mice, Knockout ; Orthomyxoviridae Infections/pathology* ; Proteolysis ; RAW 264.7 Cells ; Signal Transduction/immunology* ; THP-1 Cells ; TNF Receptor-Associated Factor 3/immunology* ; Ubiquitination/immunology* ; Viral Proteins/immunology*

OBJECTIVETo explore the effect of MiR-146a regulator function on the inflammatory response in neuroglia cell (microglia).

METHODSBV2 cells were transfected by MiR-146a mimics,and then stimulated by lipopolysaccharide (LPS). MiR-146a expression was measured by real-time polymerase chain reaction (real-time PCR). Interleukin (IL)-6 and tumor necrosis factor α (TNFα) were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) were detected by PCR and Western blotting.

RESULTSCompared to the normal control group, MiR-146a expression was significantly elevated by transfection with MiR-146a mimics (t=5.846, P=0.0021). The expression levels of IRAK1, TRAF6, TNFα, and IL-6 significantly increased in the LPS-stimulated BV2 cells compared to the non-stimulated BV2. The enhancement of MiR-146a resulted in significantly decreased IL-6 (t=5.200, P=0.0003) and TNFα (t=9.812, P<0.0001) secretion. The mRNA (t=5.353, P=0.0007) and protein (t=6.980, P=0.0009) levels of TRAF6, but not IRAK1, also significantly decreased.

CONCLUSIONMiR-146a may negatively suppress the inflammatory response of BV2 cells by regulating the expression of IRAF6 molecules in the TLR4 signaling pathway.

Blotting, Western ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Humans ; Inflammation ; Interleukin-1 Receptor-Associated Kinases ; Interleukin-6 ; Lipopolysaccharides ; MicroRNAs ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; TNF Receptor-Associated Factor 6 ; Transfection ; Tumor Necrosis Factor-alpha

OBJECTIVETo study the effect of Buyang Huanwu Decoction (BHD), Xuefu Zhuyu Decoction (XZD), and Sijunzi Decoction (SD) contained serums on expressions of Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signals, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and to explore possible anti-atherosclerotic mechanisms.

METHODSTwenty New Zealand rabbits were divided into 4 groups at random, i.e., the normal control group, the BHD group (6.7 g/kg), the XZD group (3.6 g/kg), and the SD group (1.6 g/kg), 5 in each group. All medication lasted for 7 successive days. Two h after the final medication, about 50 mL blood was withdrawn from rabbit heart for preparing serums. Human umbilical vein endothelial cell ECV304 were cultured in vitro for 18 h and randomly divided into the blank control group, the model group, the Western medicine (WM) control group, the BHD group, the XZD group, and the SD group at random. ECV304, except in the blank control group, were stimulated with lipopolysaccharide (LPS) for 2 h. Those in the WM control group and CM groups were treated respectively with corresponding CM contained serum for 24 h. Finally gene and protein expressions of TLR4, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor-6 (TRAF-6), NF-κB, LOX-1, TNF-α, ICAM-1, and VCAM-1 were detected by fluorescent quantitative PCR and Western blot.

RESULTSCompared with the blank control group, mRNA expressions of TLR4, MyD88, TRAF-6, NF-KB, LOX-1 , TNF-cx, ICAM-1, and VCAM-1 increased significantly; protein expressions of TLR4, NF-κB, LOX-1, TNF-α, ICAM-1, and VCAM-1 also increased significantly in the model group (P < 0.01). Compared with the model group, mRNA and protein expressions of each index could be significantly inhibited in the BHD group, the XZD group, and the WM control group (P < 0.05). Besides, mRNA and protein expressions of each index could be significantly elevated more in the BHD group and the XZD group than in the WM control group (P < 0.05). No statistical difference existed in each index between the SD group and the rest groups (P > 0.05).

CONCLUSIONSThe mechanism of BHD and XZD for fighting against atherosclerosis might be associated with inhibiting TLR4/NF-κB signal transduction pathway and expressions of its downstream inflammatory factors such as LOX-1, TNF-α, ICAM-1, and VCAM-1. But SD showed no associated effect on atherosclerosis.

Animals ; Atherosclerosis ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endothelial Cells ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipopolysaccharides ; Myeloid Differentiation Factor 88 ; metabolism ; NF-kappa B ; metabolism ; Rabbits ; Scavenger Receptors, Class E ; Signal Transduction ; TNF Receptor-Associated Factor 6 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Umbilical Veins ; Vascular Cell Adhesion Molecule-1 ; metabolism

Endothelial dysfunction induced by intermittent hypoxia (IH) participates in obstructive sleep apnea syndrome (OSAS)-associated cardiovascular disorders. Myeloid differentiation primary response 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) regulate numerous downstream adaptors like mitogen-activated protein kinases (MAPKs) and the subsequent oxidative stress and inflammatory responses. This study aimed to characterize the role of MyD88/TRAF6 in IH-treated cell function and its associated signaling. Human umbilical vein endothelial cells (HUVECs) were randomly exposed to IH or normoxia for 0, 2, 4 and 6 h. Western blotting was used to detect the expression pattern of target gene proteins [angiotensin 1 receptor (AT1R), p-ERK1/2, p-p38MAPK, MyD88 and TRAF6], and the relationships among these target genes down-regulated by the corresponding inhibitors were studied. Finally, the influence of these target genes on proliferation of HUVECs was also assessed by EdU analysis. Protein levels of AT1R, TRAF6 and p-ERK1/2 were increased after IH exposure, with a slight rise in MyD88 and a dynamic change in p-p38MAPK. The down-regulation of TRAF6 by siRNA reduced ERK1/2 phosphorylation during IH without any effects on AT1R. Blockade of AT1R with valsartan decreased TRAF6 and p-ERK1/2 protein expression after IH exposure. ERK1/2 inhibition with PD98059 suppressed only AT1R expression. IH promoted HUVECs proliferation, which was significantly suppressed by the inhibition of TRAF6, AT1R and ERK1/2. The findings demonstrate that TRAF6 regulates the proliferation of HUVECs exposed to short-term IH by modulating cell signaling involving ERK1/2 downstream of AT1R. Targeting the AT1R-TRAF6-p-ERK1/2 signaling pathway might be helpful in restoring endothelial function.
Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Gene Expression Regulation ; Human Umbilical Vein Endothelial Cells ; physiology ; Humans ; MAP Kinase Signaling System ; drug effects ; Phosphorylation ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Valsartan ; pharmacology

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Most cases are sporadic and its etiology is incompletely understood. However, increasing evidence suggests that oxidative stress and mitochondrial dysfunction may be involved in the pathogenesis of Parkinson's disease. The aim of this study was to investigate changes in mitochondrial protein profiles during dopaminergic neuronal cell death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were found to be significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). Among several identified candidates, TNF receptor-associated protein 1 (TRAP1), a mitochondrial molecular chaperone, was released from the mitochondria into the cytosol in MN9D cells as well as primary cultures of dopaminergic neurons following 6-OHDA treatment. This event was drug-specific in that such apoptotic inducers as staurosporine and etoposide did not cause translocation of TRAP1 into the cytosol. To our knowledge, the present study is the first to demonstrate the drug-induced subcellular translocation of TRAP1 during neurodegeneration. Further studies delineating cellular mechanism associated with this phenomenon and its functional consequence may provide better understanding of dopaminergic neurodegeneration that underlies PD pathogenesis.
Cell Death ; Cytosol ; Dopaminergic Neurons ; Electrophoresis, Gel, Two-Dimensional ; Etoposide ; Mass Spectrometry ; Mitochondria* ; Mitochondrial Proteins ; Molecular Chaperones ; Necrosis* ; Neurodegenerative Diseases ; Oxidative Stress ; Oxidopamine* ; Parkinson Disease ; Proteomics ; Staurosporine ; Substantia Nigra ; TNF Receptor-Associated Factor 1

The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.
Caspases ; metabolism ; Endopeptidases ; deficiency ; genetics ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Receptors, Antigen, T-Cell ; metabolism ; Signal Transduction ; Sumoylation ; TNF Receptor-Associated Factor 6 ; metabolism

OBJECTIVE: To construct the RNAi targeting tumor necrosis factor receptor associated factor (TRAF1) gene, and to explore the effect of interference targeting TRAF1 on the biological behavior of gastric cancer cells. METHODS: We detected the expression of TRAF1 in BGC823, SGC7901, and MGC803 gastric cancer cell lines through the real-time PCR and Western blot; then we constructed three pLVXshRNA- TRAF1-shRNAs expression vector targeting TRAF1. When TRAF1 was interfered successfully, we selected the strongest interference efficiency ShRNA by real-time PCR and Western blot. Based on interference targeting TRAF1 on gastric cancer, we tested the cell proliferation activity and apoptosis through MTT assay and flow cytometry, and the cell migration by transwell migration assay. RESULTS: The expression of TRAF1 was increased in BGC823, SGC7901, and MGC803 gastric cancer cell lines compared with gastric epithelial cells (P<0.05), and the highest expression was in BGC823 gastric cell line. In the three TRAF1 shRNAs, the strongest interference efficiency shRNA was pLVX-shRNA-TRAF1-shRNA2. When the gene TRAF1 of BGC823 was interfered, the cell growing power was weakened and the apoptosis rate increased, and the cell migration had no difference. CONCLUSION The expression of TRAF1 is up-regulated in gastric cancer cell lines BGC823, SGC7901, and MGC803, and the most obvious one is BGC823. The interference targeting TRAF1 can successfully inhibit the expression of TRAF1 in gastric cancer cell line BGC823. TRAF1 can inhibit the apoptosis of BGC823 cells.
Apoptosis ; genetics ; Base Sequence ; Cell Line, Tumor ; Humans ; Molecular Sequence Data ; RNA Interference ; RNA, Small Interfering ; genetics ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; TNF Receptor-Associated Factor 1 ; genetics ; metabolism ; Transfection ; Up-Regulation ; genetics

OBJECTIVETo investigate the function of tumor necrosis factor receptor-associated factor 1 (TRAF1) and CD30-TRAF1 signaling in Hodgkin's lymphoma.

METHODSEndogenous and CD30 ligand-induced TRAF1 expression at mRNA and protein levels were examined by quantitative RT-PCR and Western blot analyses, respectively. RNA interference was performed to silence the expression of TRAF1 in L428 cells and examine its effect on cell survival. ELISA-based NF-kappaB family transcription factor activity assay was performed to quantify the kappaB DNA-binding activity in nuclear extracts. The expression of JunB was measured by Western blot.

RESULTSTRAF1 expression was detected at both mRNA and protein levels in B cell-derived lymphoma cell lines (L428 and KM-H2). CD30 activation via binding to CD30 ligand induced the TRAF1 expression, the relative mRNA expression was increased to 7.26 +/- 0.23 from 3.50 +/- 0.20, the relative protein expression was increased to 4.53 +/- 0.55 from 2.31 +/- 0.35. The apoptosis rate was increased to (27.7 +/- 5.8)% in TRAF1-silenced L428 cells compared to (5.7 +/- 1.2)% in control cells. The p50 and RelA DNA-binding activity were decreased in TRAF1-silenced L428 cells. The expression of JunB upon CD30 ligand stimulation was not changed in TRAF1-silenced L428 cells.

CONCLUSIONSTRAF1 is overexpressed in B cell-derived Hodgkin's lymphoma cells, which is regulated by CD30 signaling pathway. TRAF1 is a crucial molecule mediating the activation of the classical NF-kappaB activity, which further facilitates the anti-apoptosis.

Apoptosis ; Cell Line, Tumor ; Hodgkin Disease ; Humans ; NF-kappa B ; metabolism ; Signal Transduction ; genetics ; TNF Receptor-Associated Factor 1