Purple tea water extract blocks RANKL-induced osteoclastogenesis through modulation of Akt/GSK3ß and Blimp1-Irf8 pathways.

A number of studies demonstrated that some tea extracts exert inhibitory effects on osteoclastogenesis induced by receptor activator of nuclear factor κB ligand (RANKL). However, the effect of purple tea, a famous tea in China, on osteoclastogenesis remains unclear. In this study, a water-based purple tea extract (PTE) was found to suppress osteoclast formation, osteoclastic resorption pit area formation, and F-actin ring formation within RANKL-stimulated bone marrow macrophages (BMMs). Furthermore, our results demonstrated that PTE could inhibit expression of master transcription factors NFATc1 and c-Fos and their target genes DC-STAMP, Ctsk, and Atp6v0d2. Western blot analysis revealed that PTE treatment led to reduced RANKL-induced phosphorylation of Akt and GSK3ß without altering transient activation of NF-κB and MAPKs (p38, JNK, ERK1/2) signaling. In addition, the results demonstrated that PTE treatment of RANKL-stimulated BMMs could down-regulate Blimp1 expression and up-regulate Irf8 expression. In summary, these results suggest that PTE treatment of RANKL-stimulated BMMs inhibited osteoclast differentiation via modulation of Blimp1-Irf8 and Akt/GSK3ß signaling pathways. Aligning with our in vitro results, in vivo PTE administration ameliorated bone loss in LPS-treated mice. Taken together, the results presented in this work suggest that PTE treatment possesses anti-osteolytic activity.

Proteomics of post-translational modifications in colorectal cancer: Discovery of new biomarkers.

Colorectal cancer (CRC) is one of the costliest health problems and ranks second in cancer-related mortality in developed countries. With the aid of proteomics, many protein biomarkers for the diagnosis, prognosis, and precise management of CRC have been identified. Furthermore, some protein biomarkers exhibit structural diversity after modifications. Post-translational modifications (PTMs), most of which are catalyzed by a variety of enzymes, extensively increase protein diversity and are involved in many complex and dynamic cellular processes through the regulation of protein function. Accumulating evidence suggests that abnormal PTM events are associated with a variety of human diseases, such as CRC, thus highlighting the need for studying PTMs to discover both the molecular mechanisms and therapeutic targets of CRC. In this review, we begin with a brief overview of the importance of protein PTMs, discuss the general strategies for proteomic profiling of several key PTMs (including phosphorylation, acetylation, glycosylation, ubiquitination, methylation, and citrullination), shift the emphasis to describing the specific methods used for delineating the global landscapes of each of these PTMs, and summarize the recent applications of these methods to explore the potential roles of the PTMs in CRC. Finally, we discuss the current status of PTM research on CRC and provide future perspectives on how PTM regulation can play an essential role in translational medicine for early diagnosis, prognosis stratification, and therapeutic intervention in CRC.

Sec-O-Glucosylhamaudol Inhibits RANKL-Induced Osteoclastogenesis by Repressing 5-LO and AKT/GSK3ß Signaling.

Sec-O-glucosylhamaudol (SOG), an active flavonoid compound derived from the root of Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., exhibits analgesic, anti-inflammatory, and high 5-lipoxygenase (5-LO) inhibitory effects. However, its effect on osteoclastogenesis was unclear. We demonstrated that SOG markedly attenuated RANKL-induced osteoclast formation, F-actin ring formation, and mineral resorption by reducing the induction of key transcription factors NFATc1, c-Fos, and their target genes such as TRAP, CTSK, and DC-STAMP during osteoclastogenesis. Western blotting showed that SOG significantly inhibited the phosphorylation of AKT and GSK3ß at the middle-late stage of osteoclastogenesis without altering calcineurin catalytic subunit protein phosphatase-2ß-Aα expression. Moreover, GSK3ß inhibitor SB415286 partially reversed SOG-induced inhibition of osteoclastogenesis, suggesting that SOG inhibits RANKL-induced osteoclastogenesis by activating GSK3ß, at least in part. 5-LO gene silencing by small interfering RNA in mouse bone marrow macrophages markedly reduced RANKL-induced osteoclastogenesis by inhibiting NFATc1. However, it did not affect the phosphorylation of AKT or GSK3ß, indicating that SOG exerts its inhibitory effects on osteoclastogenesis by suppressing both the independent 5-LO pathway and AKT-mediated GSK3ß inactivation. In support of this, SOG significantly improved bone destruction in a lipopolysaccharide-induced mouse model of bone loss. Taken together, these results suggest a potential therapeutic effect for SOG on osteoclast-related bone lysis disease.

The potential of medicinal plant extracts in improving the phytoremediation capacity of Solanum nigrum L. for heavy metal contaminated soil.

This study focused on the effects of eight medicinal plant extracts on Solanum nigrum L. potential to accumulate Cd and Pb from soil. These medicinal plants were common and relatively cheap. The eight 10% water extracts were made from the peel of Citrus reticulata Blanco (PCR), fruit of Phyllanthus emblica L. (FPE), root of Pueraria Lobata (Willd.) Ohwi (RPL), rhizome of Polygonatum sibiricum Red (RPS), root of Astragalus propinquus Schischkin (RAP), bud of Hemerocallis citrina Baroni (BHC), seed of Nelumbo nucifera Gaertn (SNN) and fruit of Prunus mume (Sieb.) Sieb.etZuce (FPM). The results showed that among all exposures, the treatment with FPE resulted in the significant increase (p < 0.05) of Cd and Pb concentration in shoots and roots of S. nigrum by 32.5% and 65.2% for Cd, and 38.7% and 39.6% for Pb. The biomasses of S. nigrum in all plant extract treatments were not significantly changed (p < 0.05) compared to the control (CK). The Cd and Pb extraction rates of S. nigrum in FPE treatment were increased respectively by 60.5% and 40.5% compared to CK. Though the treatment with EDTA significantly improved (p < 0.05) the concentration of Cd and Pb of S. nigrum, the Cd and Pb masses (ug plant-1) of S. nigrum did not show any significant difference compared to the CK due to the significant decrease in the shoot (20.4%) and root (22.0%) biomasses. The chelative role of FPE might be relation with its higher polyphenolic compounds. However, not sure if the contents of polyphenolic compounds was the only differences between FPE and other additives. Thus, some unknown organic matters might also play active role. This study provided valuable information on improving the phytoremediation potential of hyperaccumulator.

Agrimophol suppresses RANKL-mediated osteoclastogenesis through Blimp1-Bcl6 axis and prevents inflammatory bone loss in mice.

Excessive activity of osteoclasts causes many bone-related diseases, such as rheumatoid arthritis and osteoporosis. Agrimophol (AGR), a phenolic compound, originated from Agrimonia pilosa Ledeb. In prior studies, AGR is reported to possess schistosomicidal and mycobactericidal activities. However, no reports covered its anti-osteoclastogenesis characteristic. In this study, we found that AGR inhibited RANKL-induced osteoclastogenesis, bone-resorption, F-actin ring formation, and the mRNA expression of osteoclast-associated genes such as CTSK, TRAP, MMP-9, and ATP6v0d2 in vitro. In addition, AGR suppressed RANKL-induced expression of c-Fos and NFATc1. However, AGR treatment did not affect NF-κB activation and MAPKs phosphorylation in RANKL-stimulated BMMs, which implicated that AGR might not influence the initial expression of NFATc1 mediated by NF-κB and MAPKs signaling. Our results further indicated that AGR did not alter phosphorylation levels of GSK3ß and the expression of calcineurin, which implicated that AGR treatment might not interfere with phosphorylation and de-phosphorylation of NFATc1 mediated by GSK3ß and calcineurin, respectively. B-lymphocyte-induced maturation protein-1 (Blimp1), which was regarded as a transcriptional repressor of negative regulators of osteoclastogenesis, was markedly attenuated in the presence of AGR, leading to the enhanced expression of B-cell lymphoma 6 (Bcl-6). Meanwhile, Blimp1 knockdown in BMMs by siRNA strongly enhanced the expression of Bcl6 and reduced NFATc1 induction by RANKL. These findings suggested that AGR inhibited RANKL-induced osteoclast differentiation through Blimp1-Bcl-6 signaling mediated modulation of NFATc1 and its target genes. Consistent with these in vitro results, AGR exhibited a protective influence in an in vivo mouse model of LPS-induced bone loss by suppressing excessive osteoclast activity and attenuating LPS-induced bone destruction. Hence, these results identified that AGR could be considered as a potential therapeutic agent against bone lysis disease.

Highly Efficient Peptide-Based Click Chemistry for Proteomic Profiling of Nascent Proteins.

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been widely used in a variety of scientific research, including dynamic proteomics. The current well-established protocols of CuAAC for proteomics analysis introduce labeling tags (azide- or alkyne-containing reagents) at the protein level, followed by downstream analysis by mass spectrometry. In the present study, a new method for proteomic profiling of nascent proteins relying on highly efficient peptide-based click chemistry is proposed, in which the CuAAC reaction was performed at the peptide level, leading to a significant increase in efficiency of the click conjugation reaction. A remarkable improvement in identification rate for spectrum, distinct peptide, and protein was achieved when proteins to be analyzed were proteolytically cleaved into peptides prior to the click conjugation reaction, which would be beneficial to downstream proteomics analysis, especially for the detection of AHA-tagged proteins in very low amounts.

Maqui berry exhibited therapeutic effects against DSS-induced ulcerative colitis in C57BL/6 mice.

Maqui berry (Aristotelia chilensis) is an edible berry. The study aimed to explore the therapeutic effect of maqui berry on inflammatory bowel disease. Maqui berry water extract was separated by multiple solvents extraction. The chemical bases, antioxidant and anti-inflammatory properties of different extract fractions were then compared. Dextran sodium sulfate (DSS)-induced ulcerative colitis mice were used for the pharmacological activity test in vivo. Experimental results showed that the ethyl acetate fraction of maqui berry water extract (MWE) was rich in phenols and exhibited good antioxidant and anti-inflammatory activities. MWE considerably reduced the expression of COX2 and IL-6 in LPS-stimulated RAW 264.7 cells. Inflammatory bowel disease index, MDA, NO, i-NOS, and COX2 in colon tissues and MPO, TNF-α, and IL-1ß in blood serums were remarkably decreased in the treatment group compared to in the model group (p < 0.05). Intestinal histopathological damage was significantly alleviated in the treatment group, and the expression of occludin was increased (p < 0.05). MWE treatment alleviated the imbalance of gut microbiota caused by DSS injury. Overall, MWE plays a therapeutic role in ulcerative colitis through its anti-inflammatory effect, reduces immune stress, and regulates gut microbiota.

Tatarinan N inhibits osteoclast differentiation through attenuating NF-κB, MAPKs and Ca2+-dependent signaling.

Osteoclasts are multinucleated cells that originate from hemopoietic stem cells. Targeting over activated osteoclasts is thought to be an effective therapeutic approach to osteoporosis. In a previous study, we reported that Tatarinan O, a lignin-like compound, suppressed RANKL-induced osteoclastogenesis. In this study, we further examined the effects on osteoclast formation of three lignin-like compounds including Tatarinan N (TN), Tatarinan U (TU) and Tatarinan V (TV), all containing a common structure of asarone. We found that only TN suppressed RANKL-induced osteoclast differentiation, bone resorption pit formation and F-acting ring formation. TU and TV did not influence RANKL-induced osteoclastogenesis. We also found that TN dose-dependently inhibited the expression of osteoclastogenesis-associated genes, including TRAP, cathepsin K and MMP-9. Furthermore, we found that TN down-regulated the key transcription factor NFATc1 and c-Fos by preventing the activation of NF-κB and phosphorylation of MAPKs including ERK1/2 and p38 but not JNK. TN attenuated calcineurin expression via suppression of the Btk-PLCγ2 cascade and reduction of intracellular Ca2+, modulating NFATc1 activation. Taking together, our results indicated that TN might have therapeutic potential for osteoporosis.

Procyanidin B2 mitigates behavioral impairment and protects myelin integrity in cuprizone-induced schizophrenia in mice.

Numerous studies have suggested that neuropathological changes in schizophrenia may be related to damage to white matter or demyelination. Procyanidin B2, which is a constituent of many fruits such as grapes and strawberries, has various biological activities such as anti-inflammatory and anti-tumor activity, as has been reported. This study aimed to estimate the effects of procyanidin B2 on behavioral impairment and the protection of myelin integrity in a cuprizone-induced schizophrenia model. Mice were exposed to cuprizone (0.2% w/w in chow) for five weeks to induce schizophrenia-like behavioral changes and demyelination. Procyanidin B2 (20 or 100 mg kg-1 day-1) or vehicle was administered orally to mice after withdrawal from cuprizone. Behavioral impairment was detected with an open-field test, a rotarod test and a Morris water maze. Myelin integrity was assessed using LFB staining and MBP expression, including immunofluorescence and western blotting. In addition, enhancements in the expression of HO-1 and NQO1 suggested that procyanidin B2 may regulate oxidative homeostasis via promoting the translation of Nrf2 to the nucleus. Data indicated that procyanidin B2 could mitigate behavioral impairment and protect myelin integrity in the cuprizone-induced model via regulating oxidative stress by activating Nrf2 signaling.

Sciadopitysin suppresses RANKL-mediated osteoclastogenesis and prevents bone loss in LPS-treated mice.

Previous studies reported that sciadopitysin (Sc), a type of biflavonoids, protects reactive oxygen species (ROS)-mediated osteoblast dysfunction, but its role in osteoclastogenesis remains unclear. In this study, we observed that Sc dose-dependently suppressed RANKL-induced osteoclastogenesis and bone resorption. Our results indicated that Sc treatment strongly reduced RANKL-induced osteoclast-specific genes expression, including cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP) and MMP-9. Furthermore, Sc apparently attenuated RANKL-increased expressions of c-Fos and NFATc1. Meanwhile, Sc also strikingly inhibited the activation of NF-κB without altering the phosphorylation of MAPKs (p38, JNK and ERK1/2). Finally, our study demonstrated that Sc administration could reverse the bone loss in LPS-induced mice model. This study suggests that Sc inhibits RANKL-induced osteoclastogenesis and bone loss by inhibiting NF-κB activation and reducing the expression of c-Fos and NFATc1. Therefore, Sc might be benefit for RANKL-mediated osteolytic bone diseases.

Activation of thromboxane A2 receptors mediates endothelial dysfunction in diabetic mice.

BACKGROUND: Diabetes is one of high-risk factors for cardiovascular disease. Improvement of endothelial dysfunction in diabetes reduces vascular complications. However, the underlying mechanism needs to be uncovered. This study was conducted to elucidate whether and how thromboxane A2 receptor (TPr) activation contributes to endothelial dysfunction in diabetes. METHODS AND RESULTS: Exposure of human umbilical vein endothelial cells (HUVECs) to either TPr agonists, two structurally related thromboxane A2 (TxA2) mimetics, significantly reduced phosphorylations of endothelial nitric oxide synthase (eNOS) at Ser1177 and Akt at Ser473. These effects were abolished by pharmacological or genetic inhibitors of TPr. TPr-induced suppression of eNOS and Akt phosphorylation was accompanied by upregulation of PTEN (phosphatase and tension homolog deleted on chromosome 10) and Ser380/Thr382/383 PTEN phosphorylation. PTEN-specific siRNA restored Akt-eNOS signaling in the face of TPr activation. The small GTPase, Rho, was also activated by TPr stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase (ROCK) inhibitor, rescued TPr-impaired Akt-eNOS signaling. In mice, streptozotocin-induced diabetes was associated with aortic PTEN upregulation, PTEN-Ser380/Thr382/383 phosphorylation, and dephosphorylation of Akt (at Ser473) and eNOS (at Ser1177). Importantly, administration of TPr antagonist blocked these changes. CONCLUSION: We conclude that TPr activation impairs endothelial function by selectively inactivating the ROCK-PTEN-Akt-eNOS pathway in diabetic mice.

MicroRNA-125b-5p suppresses Brucella abortus intracellular survival via control of A20 expression.

BACKGROUND: Brucella may establish chronic infection by regulating the expression of miRNAs. However, the role of miRNAs in modulating the intracellular growth of Brucella remains unclear. RESULTS: In this study, we show that Brucella. abortus infection leads to downregulation of miR-125b-5p in macrophages. We establish that miR-125b-5p targets A20, an inhibitor of the NF-kB activation. Additionally, expression of miR-125b-5p decreases A20 expression in B. abortus-infected macrophages and leads to NF-kB activation and increased production of TNFα. Furthermore, B. abortus survival is attenuated in the presence of miR-125b-5p. CONCLUSIONS: These results uncover a role for miR-125b-5p in the regulation of B. abortus intracellular survival via the control of A20 expression.

The role of silicon in enhancing resistance to bacterial blight of hydroponic- and soil-cultured rice.

Here we report for the first time that bacterial blight of rice can be alleviated by silicon (Si) added. In both inoculated and uninoculated plants, shoot dry weight was significantly higher in the +Si plants than in the -Si plants. A soil-cultured trial showed that disease severity was 24.3% lower in the Si-amended plants than in the non-Si-amended plants. Plants that were switched from -Si to +Si nutrient solution and simultaneously inoculated with Xoo also exhibited the same high resistance to bacterial blight as the plants that were treated continuously with Si, with control efficiencies of 52.8 and 62.9%, respectively. Moreover, total concentrations of soluble phenolics and lignin in rice leaves were significantly higher in the +Si plants than in the -Si plants. Polyphenoloxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities in rice leaves were observed to be higher in the +Si plants than in the -Si plants. The expression levels of Os03g0109600, Prla, Rcht2 and Lox2osPil, were also higher in +Si plants than in -Si plants post-inoculation during the experimental time. Addition of Si resulted in increased Pal transcription, and inhibited CatA and Os03g0126000 expression in the earlier and later stages of bacterial inoculation, respectively.

Tatarinan O, a lignin-like compound from the roots of Acorus tatarinowii Schott inhibits osteoclast differentiation through suppressing the expression of c-Fos and NFATc1.

Osteoclasts (OC) are large multinucleated cells derived from monocyte/macrophage precursors. Suppressing osteoclastogenesis is considered as an effective therapeutic approach to erosive bone disease. The root of Acorus tatarinowii Schott, a well-known traditional Chinese medicine was used to treat rheumatosis and other inflammatory disease. However, the effects of tatarinan O (TO), one of the lignin-like compounds isolated from the roots of Acorus tatarinowii Schott during bone development are still unclear. In the present study, we explored the effect of TO on RANKL-induced osteoclastogenesis in vitro. TO was found to suppress osteoclast differentiation from RANKL-stimulated mouse bone marrow macrophages (BMMs) without significant cytotoxicity. TO also dose-dependently suppressed bone resorption activity of mature osteoclasts. Additionally, TO apparently inhibited the expression of osteoclastic marker genes, such as MMP-9, Cts K and TRAP. Furthermore, our results showed that TO decreased RANKL-induced expression of c-Fos and NFATc1 without influencing NF-κB activation and MAPK phosphorylation. Hence, for the first time we revealed that TO dose-dependently inhibited osteoclastogenesis from RANKL-stimulated mouse BMMs via decreasing the expression of NFATc1 and c-Fos.

Inositol-Requiring Enzyme 1-Dependent Activation of AMPK Promotes Brucella abortus Intracellular Growth.

UNLABELLED: AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process of Brucella infection remains unknown. Our data demonstrate that B. abortus infection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKß, and TAK1 are not required for the activation of AMPK by B. abortus infection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found that B. abortus infection-induced IRE1-dependent activation of AMPK promotes B. abortus intracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production. IMPORTANCE: Previous studies showed that B. abortus infection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused by B. abortus infection plays important role in NADPH oxidase-derived ROS production.

The Protective Effects of HJB-1, a Derivative of 17-Hydroxy-Jolkinolide B, on LPS-Induced Acute Distress Respiratory Syndrome Mice.

Acute respiratory distress syndrome (ARDS),which is inflammatory disorder of the lung, which is caused by pneumonia, aspiration of gastric contents, trauma and sepsis, results in widespread lung inflammation and increased pulmonary vascular permeability. Its pathogenesis is complicated and the mortality is high. Thus, there is a tremendous need for new therapies. We have reported that HJB-1, a 17-hydroxy-jolkinolide B derivative, exhibited strong anti-inflammatory effects in vitro. In this study, we investigated its impacts on LPS-induced ARDS mice. We found that HJB-1 significantly alleviated LPS-induced pulmonary histological alterations, inflammatory cells infiltration, lung edema, as well as the generation of inflammatory cytokines TNF-α, IL-1ß and IL-6 in BALF. In addition, HJB-1 markedly suppressed LPS-induced IκB-α degradation, nuclear accumulation of NF-κB p65 subunit and MAPK phosphorylation. These results suggested that HJB-1 improved LPS-induced ARDS by suppressing LPS-induced NF-κB and MAPK activation.

Calycosin Suppresses RANKL-Mediated Osteoclastogenesis through Inhibition of MAPKs and NF-κB.

Calycosin, an isoflavonoid phytoestrogen, isolated from Radix Astragali, was reported to possess anti-tumor, anti-inflammation, and osteogenic properties, but its impact on osteoclast differentiation remains unclear. In this study, we examined the effects of calycosin on osteoclastogenesis induced by RANKL. The results showed that calycosin significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). Calycosin also dose-dependently suppressed the formation of bone resorption pits by mature osteoclasts. In addition, the expression of osteoclatogenesis-related genes, including cathepsin K (CtsK), tartrate-resistant acid phosphatase (TRAP), and MMP-9, was significantly inhibited by calycosin. Furthermore, the results indicated that calycosin down-regulated the expression levels of NFATc1 and c-Fos through suppressing the activation of NF-κB and MAPKs. Our results indicate that calycosin has an inhibitory role in the bone loss by preventing osteoclast formation, as well as its bone resorptive activity. Therefore, calycosin may be useful as a therapeutic reagent for bone loss-associated diseases.

Proteomics Analysis of Cellular Proteins Co-Immunoprecipitated with Nucleoprotein of Influenza A Virus (H7N9).

Avian influenza A viruses are serious veterinary pathogens that normally circulate among avian populations, causing substantial economic impacts. Some strains of avian influenza A viruses, such as H5N1, H9N2, and recently reported H7N9, have been occasionally found to adapt to humans from other species. In order to replicate efficiently in the new host, influenza viruses have to interact with a variety of host factors. In the present study, H7N9 nucleoprotein was transfected into human HEK293T cells, followed by immunoprecipitated and analyzed by proteomics approaches. A series of host proteins co-immunoprecipitated were identified with high confidence, some of which were found to be acetylated at their lysine residues. Bioinformatics analysis revealed that spliceosome might be the most relevant pathway involved in host response to nucleoprotein expression, increasing our emerging knowledge of host proteins that might be involved in influenza virus replication activities.

Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

TRAF6-mediated degradation of DOK3 is required for production of IL-6 and TNFα in TLR9 signaling.

Our previous study showed that the downstream of kinase 3 (DOK3) is degraded during macrophage stimulation with CpG. However, the underlying mechanism and role in Toll-like receptor 9 (TLR9) signaling remains elusive. In this study, we demonstrate that CpG treatment leads to ubiquitin-mediated degradation of DOK3 via interaction with an E3 ligase TNFR-associated factor 6 (TRAF6). We also identified the 27th amino acid (lysine) of DOK3 is responsible for Ly48 polyubiquitination of DOK3. Furthermore, reintroduction of DOK3 (K27R) into DOK3-deficient macrophages abolishes DOK3 degradation induced by CpG and suppresses the production of IL-6 and TNFα. More importantly, our study uncovers a novel role of an E3 ligase TRAF6, namely, TRAF6 is also able to catalyse Lys 48 polyubiquitylation of target protein except for Lys 63 polyubiquitylation.