Advertisement
Research ArticleArticle

Trim13 Potentiates Toll-Like Receptor 2–Mediated Nuclear Factor κB Activation via K29-Linked Polyubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6

Bin Huang and Suk-Hwan Baek
Molecular Pharmacology April 2017, 91 (4) 307-316; DOI: https://doi.org/10.1124/mol.116.106716

Abstract

Ubiquitination is a versatile post-translational modification involved in nuclear factor-κB (NF-κB) activation of Toll-like receptor (TLR) signaling. Here, we demonstrated that Trim13, an E3 ubiquitin ligase, is up-regulated in macrophages upon stimulation with TLR2 ligand. Knockdown of Trim13 attenuated TLR2-mediated production of cytokines/chemokines and formation of foam cells as well as activation of NF-κB. Trim13 interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6) and potentiates NF-κB activity via ubiquitination of TRAF6. Overexpression of inactive mutant (C10/13A) or really interesting new gene (RING) deletion mutant of Trim13 did not potentiate ubiquitination of TRAF6 or activation of NF-κB. These results suggest that the effects of Trim13 are dependent on its E3 ligase activity. Trim13 used K29-linked polyubiquitin chains for TRAF6 ubiquitination to promote NF-κB activity and thus potentiated activation of TLR2-mediated immune responses. Our data identify Trim13 as a positive regulator of NF-κB activation and suggest that K29-linked polyubiquitination is a specific ubiquitin-linked pattern involved in the control of TLR2 signaling.

Introduction

The toll-like receptor (TLR) family plays a pivotal role in inflammation and immunity, including innate and adaptive immune responses (Blasius and Beutler, 2010). These receptors receive and transduce signals from pathogen-associated molecules, thereby activating nuclear factor-κB (NF-κB). Upon ligand stimulation, TLR recruits the adaptor proteins myeloid differentiation primary response 88 and interleukin-1 receptor-associated kinase (IRAK)1/4, which subsequently form a complex, tumor necrosis factor receptor-associated factor 6 (TRAF6), to trigger K63 auto-polyubiquitination. TRAF6 forms a recognition signal for the recruitment of transforming growth factor-β-activated kinase 1 (TAK1)–binding protein 1 (TAB1) or TAB2/3 and activation of TAK1. TAK1 subsequently activates IκB kinase (IKK) complex, leading to phosphorylation and subsequent degradation of the IκBα. This cascade of signaling enables the NF-κB complex to translocate to the nucleus and initiate transcriptional responses (Kawai and Akira, 2007).

Ubiquitination along with phosphorylation comprises one of the most common post-transcriptional modifications of NF-κB activation of TLR signaling. Various proteins such as TRAF6, TAB2/3, NF-κB essential modifier (NEMO/IKKγ), and IκBα are ubiquitinated during these processes (Chen, 2005). NF-κB activation by ubiquitination in TLR signaling is usually regulated by the type of E3 ubiquitin ligase, chain specificity, target proteins, and deubiquitinases (Park et al., 2014). The tripartite motif (Trim) proteins are really interesting new gene (RING)-type E3 ligases that contain a RING finger, B-boxes, and coiled-coil domain. The 76 members of the Trim family can be classified into 11 subgroups according to the variable C terminus (Li et al., 2014). Despite their common domain structure, Trim proteins have distinct cellular functions, including innate immunity (Rajsbaum et al., 2014), inflammation (Uchil et al., 2013), and cancer (Hatakeyama, 2011). In addition, some Trims regulate NF-κB activity in TLR-driven pathways. NF-κB activation is negatively or positively regulated according to the type of Trim in TLR signaling. Trim21, Trim27, Trim30α, and Trim38 act negatively in TLR-mediated NF-κB activation (Zha et al., 2006; Shi et al., 2008; Zhao et al., 2012; McEwan et al., 2013), but Trim23 and Trim62 act positively (Poole et al., 2009; Uchil et al., 2013).

Trim13 is an E3 ubiquitin ligase with auto-polyubiquitination properties. Trim13, along with Trim59, comprise a subgroup containing a transmembrane domain in the C-terminal region (Lerner et al., 2007). This E3 ligase may play a role in endoplasmic reticulum-associated degradation (Lerner et al., 2007). It has been reported that Trim13 ubiquitinates L-type channels and induces proteasomal degradation (Altier et al., 2011). Trim13 enhances apoptosis through protein kinase B (Joo et al., 2011), regulates autophagy caused by endoplasmic reticulum stress (Tomar et al., 2012), affects tumor cell growth (Gatt et al., 2013), and participates in regulation of melanoma differentiation-associated antigen 5–mediated type I interferon production (Narayan et al., 2014). In addition, Trim13 plays a role in the regulation of NF-κB activity (Gatt et al., 2013; Tomar and Singh, 2014), although the reports of NF-κB are controversial. Accordingly, it is necessary to reestablish the exact function of Trim13 in NF-κB activity regulation.

We established the role of Trim13 in TLR2-mediated NF-κB activation, investigated its target protein, and studied its physiologic function. Here, we report that Trim13 participates in TLR2-induced foam cell formation through various cytokines and chemokines. In this process, Trim13 stimulates NF-κB activity through K29-linked polyubiquitination chain formation of TRAF6.

Materials and Methods

Animals.

A total of 20 male, 6-week-old C57BL6 wild-type (WT) mice of average weight were purchased from Central Laboratory Animal (Seoul, South Korea). TLR2-deficient (average weight, C57BL6 background, 6 weeks old, male) mice were kindly provided by Dr. S.J. Lee (Seoul National University, Seoul, South Korea). For the experiments, a total of 20 mice were used. Each group of mice was housed under standard conditions (temperature at 25 ± 2°C, relative humidity (55% ± 5%), 12-hour light/dark cycle, and free access to food and water). The mice were sacrificed by cervical dislocation. The study was conducted in strict accordance with the guidelines and protocols approved by the Institutional Animal Care and Use Committee of Yeungnam University College of Medicine (Daegu, South Korea; permit number: YUMC-AEC2011-007).

Antibodies and Reagents.

The antibodies used and their sources were as follows: anti-Flag, anti-Flag M2 affinity gel (Sigma-Aldrich, St. Louis, MO); anti-green fluorescent protein (GFP), anti-hemagglutinin (HA) (Cell Signaling Technology, Beverly, MA); anti-TRAF6, anti-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA). The reagents used in the study and their sources were as follows: oil red-O, oxidized low-density lipoprotein (Sigma-Aldrich); protein A/G PLUS Agarose (Santa Cruz Biotechnology); mouse TLR agonist kits including Pam3CSK4 (InvivoGen, San Diego, CA), and Lipofectamine 2000 (Invitrogen/Thermo Fisher Scientific, Waltham, MA); Amaxa reagents (Lonza, Switzerland); reverse-transcription polymerase chain reaction (RT-PCR) kits, restriction enzymes (Takara Bio, Shiga, Japan); luciferase assay kits (Promega, Madison, WI); and enhanced chemiluminescence solution (GE Healthcare, Little Chalfont, UK).

Cell Culture and Transfection.

Raw 264.7 and HEK293FT cells were purchased from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 5% CO2 and 95% O2. Cells were plated in 35 mm or 100 mm diameter plates at an appropriate density and grown overnight. The NF-κB/293/GFP-Luc transcriptional reporter cell line was purchased from System Biosciences (Mountain View, CA). Cells were transduced with a lentivector that coexpresses destabilized GFP and firefly luciferase reporter driven by the minimal cytomegalovirus promoter in conjunction with four copies of the NF-κB consensus transcriptional response element upstream of the minimal cytomegalovirus. Primary bone marrow-derived monocytes were differentiated into bone marrow-derived macrophage (BMDM) over 5 to 7 days in Dulbecco’s modified Eagle’s medium supplemented with 10% L929 cell-conditioned medium (as a source of macrophage colony-stimulating factor). Small interfering RNA (siRNA) transfection into cells was conducted using Amaxa reagents according to the manufacturer´s instructions. Plasmids were transfected in Raw 264.7 cells employing Amaxa reagents and in HEK293FT cells using Lipofectamine 2000 reagent.

Plasmid Constructs and siRNAs.

WT Trim13 was kindly provided by Dr. J.Y. Kim (Korea Hydro & Nuclear Power Co., Gyeongju, South Korea). For Flag-tagged Trim13 and its various deletion mutants, the corresponding DNA fragments were PCR amplified and cloned into the HindIII and XbaI sites of pFlag-CMV2 vector. Trim13 was also cloned downstream from a GFP tag of pEGFP-C1 vector. Site-directed mutagenesis was used to convert WT Trim13 into a catalytic mutant form by mutating cysteine 10 and 13 into alanine (C10/13A). TRAF6 was cloned downstream from a Flag tag at the NotI and BamHI sites of pFlag-CMV2 vector. HA-Ubs (WT, K29R, K48R, K63R, K29, K48, and K63) were kindly provided by Dr. Y. Ye (National Institute of Diabetes and Digestive and Kidney Diseases, U.S. National Institutes of Health, Bethesda, MD). The plasmid constructs were systematically checked based on their sequence analyses. Stealth nonspecific control and Trim13-specific siRNAs against the following target genes were designed using the Block-IT Stealth RNAi designer (Invitrogen): Trim13 (1), 5′-GAA GCU UGC AGU UAU GCA AAC CUA U-3′; Trim13 (2), 5′-TGT ATT CCT GGA ATG GTC TCC ACT T-3′; TLR2, 5′-UAA AAG GGC GGG UCA GAG UUC UCC A-3′.

Foam Cell Formation Assay.

Raw 264.7 cells were transfected with individual Trim13 siRNAs using Amaxa reagents. For lipid uptake analysis, macrophages were cultured in six-well plates, then treated with Pam3CSK4 (100 ng/ml) and oxidized low-density lipoprotein (50 μg/ml) for 24 hours. Next, cells were washed 3 times with phosphate-buffered saline, fixed with 10% formalin, and stained with oil red-O solution. Intracellular lipid droplets were then detected by light microscopy using a DIAPHOT 300 light microscope (Nikon Corporation, Tokyo, Japan). Images were recorded with an AxioCam ICc I digital camera system (Carl Zeiss, Oberkochen, Germany).

NF-κB Promoter Activity Assay.

NF-κB/293/GFP-Luc reporter cells were transfected with Trim13 plasmids using Lipofectamine 2000. The cell lysates were then assayed for luciferase activity using the luciferase reporter assay system according to the manufacturer´s protocols. Raw 264.7 cells were transfected using Amaxa regents with siRNA or DNA expressing the Trim13 proteins and plasmid DNA expressing firefly luciferase under control of the NF-κB construct. In addition, each transfection mixture contained an internal control reporter plasmid pRL-TK, which expresses Renilla luciferase for normalization of transfection efficiencies. After transfection, the cells were incubated with Pam3CSK4 for the indicated times. The cell lysates were then assayed for luciferase activity.

In Vivo Ubiquitination Assay.

For in vivo ubiquitination assay, cells harvested from 100-mm dishes were lysed in buffer (0.5% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 5 mM N-ethylmaleimide, and protease inhibitor cocktail). The lysates were then centrifuged at 15,000 rpm for 10 minutes, after incubated with 1% SDS and 1 mM dithiothreitol at 65°C for 20 minutes, after which they were diluted 10-fold with lysis buffer. The samples were subsequently boiled at 60°C for 20 minutes to break the protein-protein interactions, after which they were diluted 10-fold with lysis buffer. Next, the diluted samples were incubated with specific antibodies or α-Flag-M2 bead at 4°C for 4 hours. Finally, the precipitates were washed 5 times using RIPA buffer and denatured in SDS loading buffer at 95°C for 10 minutes.

Western Blot Analysis.

After SDS-PAGE, the proteins were transferred to nitrocellulose membranes that were subsequently blocked with Tris-buffered saline containing 5% skim milk and 0.1% Tween20 for 1 hour at room temperature. After blocking, the membranes were incubated with primary antibodies overnight at 4°C. After washes, the membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (anti-mouse or anti-rabbit 1:10,000) for 1 hour at room temperature and visualized using an enhanced chemiluminescence solution.

Coimmunoprecipitation.

Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 5 mM EDTA, 1 mM EGTA, 10 mM NaF, 10% glycerol, and 1% Triton X-100) by incubation on ice for 30 minutes followed by centrifugation at 15,000 rpm for 10 minutes. Lysates were incubated with α-Flag-M2 affinity gel at 4°C for overnight. The precipitates were washed 5 times using lysis buffer and analyzed by immunoblotting.

RT-PCR and Quantitative Real-Time PCR.

Total RNA was extracted from cells using TRIzol. First-strand cDNA was then synthesized from 1 μg total RNA by employing random primers, oligo-dT, and reverse transcriptase (Promega). The assays of RT-PCR and quantitative real-time PCR (qPCR) were performed as described elsewhere (Lyu et al., 2015) using the primers listed in Tables 1 and 2. Target gene expression was normalized to β-actin transcription. We conducted qPCR using a LightCycler 1.5 (Roche Diagnostics, Basel, Switzerland) with SYBR-Green I as the fluorescent dye according to the manufacturer's instructions. For quantification, target genes were normalized against β-actin control and visualized using the Fuji Intelligent Dark Box LAS-3000 Image Reader (FujiFilm, Tokyo, Japan). Densitometric analysis was performed using LAS-3000 Fujifilm Image Reader and Multi Gauge 3.0 software.

View this table:
TABLE 1

RT-PCR primers used in the study

View this table:
TABLE 2

qPCR primers used in the study

Statistical Analysis.

Results are expressed as the mean ± standard deviation of a minimum of three independent assays. Statistical significance was calculated by analysis of variance using GraphPad Prism software, version 5.01 (GraphPad Software, La Jolla, CA). Groups were compared with two-way analysis of variance with a Bonferroni post-test. P < 0.05 was considered statistically significant.

Results

Trim13 Is Up-Regulated by Stimulation of TLRs.

A DNA microarray was performed under Pam3CSK4 (a TLR2 agonist) treatment in Raw 264.7 cells to identify a new regulator of the TLR2 signaling pathway. Several ubiquitin-related genes (Tnip1, RANK, A20, ABIN3, and Trim13) were included as an up-regulated gene by TLR2 activation (Tables 3 and 4). Among them, we focused on the E3 ubiquitin ligase, Trim13. We used RT-PCR to verify the expression of Trim13 via TLR2 in Raw 264.7 cells. As shown in Fig. 1A, Trim13 gene expression was induced as an early gene by Pam3CSK4 in a time-dependent manner. We also confirmed the effects of Pam3CSK4 after isolation of BMDMs from the bone marrow of WT or TLR2 knockout (KO) mice.

View this table:
TABLE 3

List of up-regulated genes in the microarray analysis

View this table:
TABLE 4

List of up-regulated genes in the microarray analysis

Fig. 1.
Fig. 1.

TLR agonists induce Trim13 gene expression. (A) Raw 264.7 cells were treated with Pam3CSK4 (100 ng/ml) for the indicated times, after which the Trim13 mRNA levels were analyzed by RT-PCR. (B) After monocytes were isolated from WT or TLR2 KO mouse bone marrow and differentiated into BMDMs, they were treated with Pam3CSK4 for the indicated times. Trim13 mRNA levels were determined by RT-PCR. (C) Raw 264.7 cells were transfected with either control or TLR2 siRNA. The transfected cells were then treated with Pam3CSK4, HKLM, LPS, or CpG after which qPCR was performed using Trim13 primers. (D) Raw 264.7 cells were treated with various TLR agonists, Pam3CSK4, HKLM, Poly I:C, LPS, flagellin, FSL1, single-stranded RNA (ssRNA), and CpG, for 40 minutes. Trim13 mRNA expression was determined by RT-PCR and qPCR. β-actin was used as a control. The representative experiments were repeated in triplicate with similar results. *P < 0.05 versus the control.

Increased Trim13 gene expression was observed in WT BMDMs treated with Pam3CSK4, whereas Pam3CSK4 had no effect on TLR2 KO BMDMs (Fig. 1B). The inducing Trim13 gene is only blocked by TLR2 ligands (Pam3CSK4 and HKLM), not TLR4 ligand (LPS) or TLR9 ligand (CpG), in the TLR2 siRNA cells (Fig. 1C).

To test the general effects of TLRs on Trim13 expression, we checked other types of TLR agonists. All tested agonists (TLR1/2, TLR2, TLR3, TLR4, TLR5, TLR6/2, TLR8, and TLR9) induced Trim13 gene expression (Fig. 1D; qPCR & RT-PCR).

We next investigated the expression pattern of Trim13 protein. To accomplish this, we used four commercially available Trim13 antibodies to detect Trim13 protein. Even though three of the four could detect Trim13 protein in the overexpression system, none of the antibodies detected endogenous or Pam3CSK4-induced Trim 13 protein in Raw 264.7 cells. Nevertheless, these data indicate that Trim13 is an important regulator of TLR signaling of macrophages, including TLR2.

Trim13 Potentiates TLR2-Mediated Macrophage Activation via NF-κB.

We attempted to identify the role of Trim13 in TLR2 signaling. It is well known that Pam3CSK4, a TLR2 ligand, activates NF-κB and then accelerates foam cell formation, eventually leading to atherosclerosis (Mullick et al., 2005). To investigate the effects of Trim13 on NF-κB activation, we constructed two types of Trim13 siRNA and then compared the effects of Pam3CSK4 in control siRNA and Trim13 siRNA transfected Raw 264.7 cells. We checked whether two types of Trim13 siRNAs knocked-down gene expression caused by TLR2 stimulation (Fig. 2A, left panel).

Fig. 2.
Fig. 2.

Trim13 regulates foam cell formation and cytokines/chemokines production via NF-κB. (A) Raw 264.7 cells were cotransfected with either control siRNA or two types of Trim13 siRNA (#1, #2) with NF-κB-Luc promoter for 24 hours. The transfected cells were then treated with Pam3CSK4 (100 ng/ml), after which qPCR was performed using Trim13 primers or samples were assayed luciferase activity. *P < 0.05 versus the control. (B, C) Raw 264.7 cells were transfected as described earlier. The siRNA transfected cells were then treated with Pam3CSK4 for 40 minutes, after which cells were harvested and the mRNA of the indicated genes was measured using qPCR (B) and RT-PCR (C). β-actin was used as a control. *P < 0.05 versus the control. (D) The siRNA transfected cells were then treated with Pam3CSK4 in the presence of oxidized low-density lipoprotein for 24 hours and stained with oil red-O solution (original magnification, 400×). The representative experiments were repeated in triplicate with similar results. IL, interleukin.

To confirm the role of Trim13, we measured the change in NF-κB activity in response to Pam3CSK4 in Raw 264.7 cells. Pam3CSK4 treatment increased NF-κB activity by 64-fold; however, Trim13 knock-down strongly attenuated the NF-κB activity induced by Pam3CSK4 (Fig. 2A, right panel). Notably, NF-κB activity was inhibited according to the level of Trim13 expression inhibition.

NF-κB is the best-known transcription factor regulating cytokines and chemokines. Therefore, we investigated expression of cytokines and chemokines using qPCR and conventional RT-PCR. Trim13 knock-down decreased expression of tumor necrosis factor α (TNF-α), interleukin-1β, and interleukin-6 as well as C-C motif chemokine ligand 2 (CCL2), CCL3, and CCL4, which are all induced by Pam3CSK4. The two types of Trim13 siRNA had inhibitory effects on cytokine and chemokine production proportional to the knock-down efficiency (Fig. 2, B and C).

Because cytokines and chemokines are essential to the formation of foam cells, we tested and compared their formation in control siRNA and Trim13 siRNA transfected cells. As shown in Fig. 2D, foam cell formation by TLR2 stimulation was dramatically decreased in Trim13 knock-down cells. Foam cell formation was also inhibited according to the level of Trim13 gene expression.

These results suggest that NF-κB activity is positively regulated by Trim13. Furthermore, these findings indicate that TLR2 stimulation by Pam3CSK4 increases Trim13 expression, thereby causing cytokines and chemokines production via NF-κB activation and eventually promoting foam cell formation.

Trim13 Interacts with and Ubiquitinates TRAF6.

We investigated the mechanism by which Trim13 increases NF-κB activation. TRAF6 is a very important adaptor protein during the initial stages of NF-κB activation induced by TLR2 (Akira and Takeda, 2004). We speculated that TRAF6 was a target of Trim13, so we studied their interaction. Trim13 and TRAF6 plasmids were cotransfected in HEK293FT cells. We detected clear binding of Trim13 with TRAF6, but not with NEMO and TAB1 (Fig. 3A).

Fig. 3.
Fig. 3.

Trim13 binds and ubiquitinates TRAF6 and activates NF-κB. (A) HEK293FT cells transfected Flag-tagged TRAF6, NEMO, or TAB1 with GFP-tagged Trim13. Total lysates were isolated from the transfected cells, immunoprecipitation was then conducted using Flag beads, and the samples were subjected to electrophoresis and immunoblotting with anti-GFP antibody. (B) HEK293FT cells were transfected with either GFP-tagged WT Trim13 or mutant (C10/13A). After 24 hours, the cells were harvested in RIPA buffer with high SDS concentrations. The samples were then diluted 10-fold with RIPA buffer, after which they were subjected to immunoprecipitation using anti-TRAF6 antibody, followed by electrophoresis and immunoblotting with anti-TRAF6 antibody. The ubiquitinated TRAF6 was detected by anti-HA antibody. (C) TRAF6 was transfected with either WT Trim13 or mutant (C10/13A) in HEK293FT cells for 24 hours. The cell lysates of transfected cells were subjected to in vivo ubiquitination assay by immunoprecipitation using Flag beads, after which the ubiquitinated TRAF6 was detected by anti-HA antibody. (D) Flag-TRAF6 was transfected with either WT Trim13 or mutant (C10/13A) in HEK293FT cells for 24 hours. Immunoprecipitation was then conducted using Flag beads, and samples were subjected to electrophoresis and immunoblotting with anti-GFP antibody. (E) HEK293FT cells were transfected with either Flag-tagged TRAF6 or NEMO with Trim13 and HA-Ub for 24 hours. The cell lysates of transfected cells were then subjected to in vivo ubiquitination assay by immunoprecipitation using Flag beads, after which the ubiquitinated TRAF6 or NEMO was detected by anti-HA antibody. (F) Flag-TRAF6 was coexpressed with either WT GFP-Trim13 or mutant (C10/13A) in NF-κB/293/GFP-Luc reporter cells for 24 hours. Luciferase activity was measured at 24 hours after transfection. *P < 0.05 versus the control. TRAF6 and Trim13 expression was determined by immunoblotting using GFP or Flag antibody. (G) Raw 264.7 cells were treated with Pam3CSK4 for the indicated times, after which TRAF6 ubiquitination was analyzed by in vivo ubiquitination assay. (H) Raw 264.7 cells were transfected with either control siRNA or Trim13 siRNA for 24 hours. The siRNA-transfected cells were then treated with Pam3CSK4 for 30 minutes, after which TRAF6 ubiquitination was analyzed by in vivo ubiquitination assay. Representative experiments were repeated in triplicate with similar results.

Regulation of NF-κB by TRAF6 is dependent on ubiquitination (Zhou et al., 2012; Zhang et al., 2013). In addition, the E3 ligase activity of Trim13 is dependent on the 10th and 13th Cys residues. Therefore, we generated a construct containing a Cys10/13 to Ala10/13 mutation (C10/13A) and then tested for ubiquitination of endogenous TRAF6 by Trim13 in cells. WT Trim13 clearly accelerated TRAF6 ubiquitination, but had no effect on the C10/13A mutant Trim13 (Fig. 3B). We also looked for ubiquitination of exogenous TRAF6 by Trim13 in these cells. WT Trim13 stimulated exogenous TRAF6 ubiquitination, similar to endogenous TRAF6, but had no effect on C10/13A mutant (Fig. 3C).

Trim13 not only plays a role in the ubiquitination of other proteins but also degrades via auto-ubiquitination. Therefore, in the input, WT and the mutant had the level of Trim13 protein to be different. However, not only the WT but also C10/13A mutant Trim13 showed binding with TRAF6 (Fig. 3D). These results suggest that Trim13 enzyme activity mediates TRAF6 ubiquitination.

A previous study suggested that Trim13 regulates tumor necrosis factor receptor (TNFR)–mediated NEMO ubiquitination (Tomar and Singh, 2014). Therefore, we investigated and compared TRAF6 and NEMO ubiquitination by Trim13. Interestingly, ubiquitination of NEMO not affected, while ubiquitination of TRAF6 was dramatically increased in HEK293FT cells coexpressing Trim13 (Fig. 3E).

We next tested NF-κB activation by the combination of Trim13 and TRAF6 using the NF-κB/293/GFP-Luc reporter cell line. Before we assayed the NF-κB activity, we checked the expression of TRAF6 and Trim13 in a transfected reporter cell line (Fig. 3F, upper panel). NF-κB activity dramatically increased in response to transfection of TRAF6 alone. NF-κB activity was also much higher in cells cotransfected with TRAF6 and WT Trim13 relative to TRAF6 transfected alone. However, cotransfection of TRAF6 and the C10/13A Trim13 mutant had similar effects as TRAF6 transfection alone (Fig. 3F, lower panel).

The endogenous ubiquitination of TRAF6 by TLR2 ligand was investigated in Raw 264.7 cells. Treatment with Pam3CSK4 increased TRAF6 ubiquitination of Raw 264.7 cells in a time-dependent manner (Fig. 3G). We confirmed that TRAF6 ubiquitination was regulated by Trim13 using siRNA. Trim13 knock-down significantly attenuated the increase in TRAF6 ubiquitination induced by Pam3CSK4 (Fig. 3H).

Overall, these results suggest that Trim13 stimulates TRAF6 ubiquitination, thereby potentiating NF-κB activity in TLR2 signaling.

Trim13 Regulates NF-κB Activity via Its E3 Ligase Activity.

Trim13 is a protein composed of multiple domains, including a RING, B-boxes, coiled coil (CC), and a transmembrane (TM) domain. Each domain has a specific function in the signaling pathway (Li et al., 2014). We constructed deletion mutants to verify the importance of each domain in NF-κB activation. The domain composition is shown in Fig. 4A.

Fig. 4.
Fig. 4.

Trim13 regulates TRAF6 ubiquitination and NF-κB activity via E3 ligase activity of the RING domain. (A) Domain organization of Trim13. (B) Flag-tagged TRAF6 was coexpressed with either GFP-tagged WT Trim13 or different deletion mutants in HEK293FT cells for an in vivo ubiquitination assay as described in the Fig. 3C caption. The protein expression of different Trim13 mutants was determined by immunoblotting using anti-GFP antibody. The protein expression of TRAF6 was determined by immunoblotting using anti-Flag antibody. (C) NF-κB/293/GFP-Luc reporter cells were transfected with the indicated plasmids of Trim13 WT or each deletion mutants with Flag-tagged TRAF6. Luciferase activity was measured at 24 hours after transfection. The representative experiments were repeated in triplicate with similar results. *P < 0.05 versus the control.

HEK293FT cells were cotransfected with each Trim13 mutant plus TRAF6 plasmids, after which TRAF6 ubiquitination was investigated. Each Trim13 mutants was successfully expressed. Overexpression of WT Trim13 increased TRAF6 polyubiquitination, but the RING deletion mutant did not have any effect, similar to the C10/13A mutant shown in Fig. 3C. Conversely, deletion of CC or TM domains showed no difference relative to the WT Trim13 effects (Fig. 4B).

We next investigated the change in NF-κB activity. The increase in NF-κB activity by WT Trim13 decreased in the RING deletion mutant, but had no effect in the CC and TM deletion mutants (Fig. 4C). These results suggest that TRAF6 polyubiquitination and subsequent NF-κB activation by Trim13 is dependent on the E3 ligase activity.

Trim13 Ubiquitinates TRAF6 via the K29-Linked Polyubiquitination Chain.

TRAF6 ubiquitination was an important mechanism in the increase of NF-κB activity by Trim13. Therefore, we analyzed the type of ubiquitin chain generated on TRAF6 by Trim13. To accomplish this, we first used specific ubiquitin mutants (K29R, K48R, and K63R) to test their effects on TRAF6 ubiquitination in cells overexpressing Trim13. Although K48R and K63R ubiquitin also showed some decrease, K29R ubiquitin showed the most inhibition of Trim13-mediated TRAF6 polyubiquitination (Fig. 5A).

Fig. 5.
Fig. 5.

Trim13 ubiquitinates TRAF6 via K29-linked polyubiquitination chain. (A) HEK293FT cells were transfected with a combination of Flag-tagged TRAF6, GFP-tagged Trim13, and various types of HA-Ub (WT or substitution of Lys with Arg [K29R, K48R, and K63R]). An in vivo ubiquitination assay was performed after 24 hours, and the expression of samples was determined by immunoblotting. (B) HEK293FT cells were transfected with a combination of specific HA-Ub mutants (WT or Lys-linked [K29, K48, and K63]), Flag-TRAF6, and GFP-Trim13 for 24 hours. The cell lysates of transfected cells were subjected to in vivo ubiquitination assay by immunoprecipitation using Flag beads, after which the ubiquitinated TRAF6 was detected by anti-HA antibody. The representative experiments were repeated in triplicate with similar results. (C), NF-κB/293/GFP-Luc reporter cells were transfected WT or ubiquitin mutants (K29 or K29R) with Flag-TRAF6 and GFP-Trim13 combination. Luciferase activity was measured at 24 hours after transfection. The representative experiments were repeated in triplicate with similar results. The representative experiments were repeated in triplicate with similar results. *P < 0.05 versus the control. IB, immunoblotting; IP, immunoprecipitation.

We next used another set of ubiquitin mutants in which all except one Lys residue was replaced by Arg. Preliminary experiments showed differences in the transfection efficiency of TRAF6 when various plasmids were cotransfected with different ubiquitin mutants. Nevertheless, K29-linked polyubiquitin chain formation was a strong candidate for TRAF6 (data not shown). Therefore, we confirmed Trim13-induced polyubiquitination of TRAF6 under the same conditions after transfecting each ubiquitin mutant.

As shown in Fig. 5B, K29-ubiquitin and K63-ubiquitin promoted chain formation on TRAF6, but the effect was more powerful when K63-ubiquitin was used. Moreover, no chain formation occurred when K48-ubiquitin was used, and Trim13 only potentiated TRAF6 poly-ubiquitination in the K29-ubiquitin assay system.

We compared WT or mutant ubiquitin to prove the importance of K29-linked chain formation in regulating NF-κB activity. As shown in Fig. 5C, WT and K29 ubiquitin increased NF-κB activity by Trim13, but K29R ubiquitin could not (Fig. 5C). Thus, our results provide evidence of K29-linked polyubiquitin chain formation on TRAF6 by the Trim13 E3 ligase.

Discussion

In this study, we conducted screening to identify a new gene regulating the immune signaling pathway using a TLR2 agonist in macrophages. Among the screened genes, we identified Trim13, a RING type ubiquitin E3 ligase. This protein promoted formation of foam cells and production of cytokines/chemokines through NF-κB. Specifically, the effects of Trim13 resulted from the formation of K29-linked polyubiquitination chains of TRAF6, and ultimately increased NF-κB activity.

The Trim family has been shown to play an important role in innate immunity (Rajsbaum et al., 2014). Notably, some Trim family members regulate anti-inflammatory or proinflammatory responses through NF-κB activation. Furthermore, there are various target proteins of Trim involved in NF-κB activity regulation. For example, Trim5α and Trim8 target TAK1 (Gong et al., 2011; Li et al., 2011), Trim27 targets IKKβ (Zha et al., 2006), Trim30α targets TAB2/3 (Shi et al., 2008), and Trim40 targets NEMO (Noguchi et al., 2011).

TRAF6 is also known as an important target regulating NF-κB activation. TRAF6 is a member of the TRAFs, which mediate TLR intracellular signaling. TRAF6 has been shown to play a critical role in NF-κB activation in that TRAF6-dominant negative cells failed to respond to TLR4 stimulation (Guillot et al., 2004). TRAF6 is also critical for various types of TLRs-mediated NF-κB activation and regulation of the subsequent proinflammatory response. In these TLR pathways, stimulation of the receptors leads to recruitment of myeloid differentiation primary response 88, which further recruits IRAK1 and IRAK4. IRAK1 then binds to TRAF6, which subsequently activates downstream signaling cascades, including those of IKK (Kawai and Akira, 2007). However, Trim38 is the only type of Trim that has been reported to regulate NF-κB activity by targeting TRAF6 in TLR-mediated immune response (Zhao et al., 2012). Therefore, our discovery of Trim13 regulating NF-κB activity by targeting TRAF6 has significant meaning.

The mechanism of Trim13 in regulation of NF-κB activity is currently the subject of debate. Some reports have shown that Trim13 increases NF-κB, while others indicate that it decreases NF-κB activity. For example, Uchil et al. (2013) reported that screening 43 types of human Trim clearly demonstrated that Trim13 increases NF-κB activity, along with Trim1, Trim5, Trim25, and Trim62. Gatt et al. (2013) and Versteeg et al. (2013) also reported that Trim13 is a NF-κB activator. However, Tomar and Singh (2014) reported that Trim13 inhibits NF-κB activity by NEMO ubiquitination in tumor necrosis factor receptor (TNFR) signaling. Therefore, it is necessary to establish the correct mechanism of Trim13 in NF-κB activity regulation.

In our present study, Trim13 was found to increase NF-κB activity, thereby stimulating cytokines and chemokines production. In addition to the increase in NF-κB activity, we believe the acceleration of foam cell formation was also important. We reached these conclusions using a used Trim13 knock-down system and confirmed the results with a Trim13 overexpression system to confirm those results. Specifically, the Pam3CSK4 effect was compared between WT and C10/13A Trim13 transfected Raw 264.7 cells. NF-κB activity by Pam3CSK4 was potentiated in WT Trim13 overexpressed cells when compared with cells transfected with only vector, while C10/13A mutant overexpressed cells had no effect (data not shown). NF-κB activity was also observed in response to the degradation of IκBα protein. We also monitored changes in the IκBα protein stimulated by Pam3CSK4 in vector or Trim13-overexpressed cells. Pam3CSK4 treatment showed a more rapid degradation of IκBα in Trim13-overexpressed cells (data not shown).

Our results were contrary to those of Tomar and Singh (2014), even though the experimental conditions were very similar. Specifically, they reported that Trim13 inhibits TNFR-mediated NF-κB activation. There are several possible reasons for this discrepancy, including the difference in TLR and TNFR could be a possible for these differences. Specifically, TLR and TNFR signaling have common signaling pathways for NF-κB activation, but unique processes for each receptor. The pathways through the TAK, IKK, and NF-κB complex are very similar. However, receptor-interacting protein 1 is an important adaptor in early signaling for TNFR, while TRAF6 is associated with TLR.

Second, the methods used to measure NF-κB promotor activity could have caused these differences. While we used the NF-κB/293/GFP-Luc reporter cell line, Tomar and Singh used transient transfection methods for NF-κB promoter activity. We also attempted transient cotransfection of the NF-κB promoter gene. Even though we compensated for the transfection efficiency by cotransfection of the Renilla luciferase gene, we switched over to the cell line system for our experiments. The usage of stable cell line for NF-κB promoter activity ruled out transfection variations in these assays.

Third, some Trim mechanisms can have opposite effects according to the target protein. For example, Trim38 acts as a negative regulator when targeting TRAF6 (Zhao et al., 2012), while it acts as a positive regulator when targeting TAB2 (Hu et al., 2014). Trim21 also acts as a negative regulator targeting IKKβ (Niida et al., 2010), while it acts as a positive regulator targeting TAK (McEwan et al., 2013). This possibility cannot be excluded for Trim13 as well.

Protein ubiquitination is a post-translational modification tagging the ubiquitin to the Lys residues of target proteins. Ubiquitin chains linked through different lysine residues have various functions (Chen and Sun, 2009). For example, K48-linked chains act as a signal by targeting substrates for proteasomal degradation. In contrast, K63-linked chains are involved in various signaling pathways. Control of TRAF6 activity also depends largely on K63-linked polyubiquitination. Hence, regulation of TRAF6 polyubiquitination is an important method of controlling NF-κB signaling. Even though the roles of K48- or K63-linked ubiquitin chains have been the most widely studied, there is increasing evidence that other types of ubiquitin chains also target substrates for proteasomal degradation (Bedford et al., 2011), protein trafficking (Yuan et al., 2014), and immune signaling (Liu et al., 2014). However, although it has been detected in yeast and mammals, the role of K29-linked ubiquitin chains is poorly understood.

Some reports have suggested the importance of K29-linked polyubiquitination. Specifically, Itch/AIP4 was shown to mediate deltex degradation by K29-linked polyubiquitin chain formation (Chastagner et al., 2006), and Smurf1 regulates Wnt/β-catenin signaling through K29-linked ubiquitin chain formation of axin (Fei et al., 2013). Recently, Kristariyanto et al. (2015) and Michel et al. (2015) suggested the existence of K29-linked polyubiquitin as well. However, no studies have investigated the K29-linked polyubiquitin chain formation of TRAF6 protein and its function. Therefore, we believe that Trim13 accelerating NF-κB activity by K29-linked polyubiquitin chain formation of TRAF6 in TLR2 signaling is an important discovery.

Overall, the results of this study showed that TLR2 stimulus increased Trim13 expression, which regulated foam cell formation after production of various cytokines and chemokines by TRAF6 K29-linked ubiquitin chain formation. This process was followed by NF-κB activation. Thus, Trim13 acts as a positive regulator of TLR2-induced NF-κB activation.

Acknowledgments

The authors thank Yihong Ye (NIH, NIDDK) for HA-ubiquitin mutant constructs, and Ji Young Kim (Korea Hydro & Nuclear Power Co., South Korea) for the Trim13 construct.

Authorship Contributions

Participated in research design: Huang, Baek.

Conducted experiments: Huang.

Performed data analysis: Huang, Baek.

Wrote or contributed to the writing of the manuscript: Huang, Baek.

Footnotes

    • Received August 29, 2016.
    • Accepted January 3, 2017.

  • This work was supported by a 2014 Yeungnam University Research Grant [Grant 214A480011].

  • dx.doi.org/10.1124/mol.116.106716.

Abbreviations

BMDM
bone marrow-derived macrophage
CC
coiled coil
CCL
C-C motif chemokine ligand
GFP
green fluorescent protein
HA
influenza hemagglutinin
IKK
IκB kinase
IRAK
interleukin-1 receptor-associated kinase
KO
knockout
NEMO
nuclear factor-κB essential modulating protein
NF-κB
nuclear factor-κB
PCR
polymerase chain reaction
qPCR
quantitative real-time PCR
RING
really interesting new gene
RIPA
radioimmunoprecipitation assay
RT-PCR
reverse-transcription polymerase chain reaction
siRNA
small interfering RNA
TAB
transforming growth factor-β-activated kinase 1–binding protein
TAK
transforming growth factor-β-activated kinase
TLR
Toll-like receptor
TM
transmembrane
TNF
tumor necrosis factor
TNFR
tumor necrosis factor receptor
TRAF6
tumor necrosis factor receptor-associated factor 6
Trim
tripartite motif
Ub
ubiquitin
WT
wild type

References

  1. ↵
    1. Akira S and
    2. Takeda K
    (2004) Toll-like receptor signalling. Nat Rev Immunol 4:499–511.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Altier C,
    2. Garcia-Caballero A,
    3. Simms B,
    4. You H,
    5. Chen L,
    6. Walcher J,
    7. Tedford HW,
    8. Hermosilla T, and
    9. Zamponi GW
    (2011) The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels. Nat Neurosci 14:173–180.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Bedford L,
    2. Layfield R,
    3. Mayer RJ,
    4. Peng J, and
    5. Xu P
    (2011) Diverse polyubiquitin chains accumulate following 26S proteasomal dysfunction in mammalian neurones. Neurosci Lett 491:44–47.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Blasius AL and
    2. Beutler B
    (2010) Intracellular toll-like receptors. Immunity 32:305–315.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Chastagner P,
    2. Israël A, and
    3. Brou C
    (2006) Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains. EMBO Rep 7:1147–1153.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Chen ZJ
    (2005) Ubiquitin signalling in the NF-κB pathway. Nat Cell Biol 7:758–765.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Chen ZJ and
    2. Sun LJ
    (2009) Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 33:275–286.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Fei C,
    2. Li Z,
    3. Li C,
    4. Chen Y,
    5. Chen Z,
    6. He X,
    7. Mao L,
    8. Wang X,
    9. Zeng R, and
    10. Li L
    (2013) Smurf1-mediated Lys29-linked nonproteolytic polyubiquitination of axin negatively regulates Wnt/β-catenin signaling. Mol Cell Biol 33:4095–4105.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Gatt ME,
    2. Takada K,
    3. Mani M,
    4. Lerner M,
    5. Pick M,
    6. Hideshima T,
    7. Carrasco DE,
    8. Protopopov A,
    9. Ivanova E,
    10. Sangfelt O, et al.
    (2013) TRIM13 (RFP2) downregulation decreases tumour cell growth in multiple myeloma through inhibition of NF kappa B pathway and proteasome activity. Br J Haematol 162:210–220.
    OpenUrl
  10. ↵
    1. Gong J,
    2. Shen XH,
    3. Qiu H,
    4. Chen C, and
    5. Yang RG
    (2011) Rhesus monkey TRIM5α represses HIV-1 LTR promoter activity by negatively regulating TAK1/TAB1/TAB2/TAB3-complex-mediated NF-κB activation. Arch Virol 156:1997–2006.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Guillot L,
    2. Medjane S,
    3. Le-Barillec K,
    4. Balloy V,
    5. Danel C,
    6. Chignard M, and
    7. Si-Tahar M
    (2004) Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. J Biol Chem 279:2712–2718.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Hatakeyama S
    (2011) TRIM proteins and cancer. Nat Rev Cancer 11:792–804.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Hu MM,
    2. Yang Q,
    3. Zhang J,
    4. Liu SM,
    5. Zhang Y,
    6. Lin H,
    7. Huang ZF,
    8. Wang YY,
    9. Zhang XD,
    10. Zhong B, et al.
    (2014) TRIM38 inhibits TNFα- and IL-1β-triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3. Proc Natl Acad Sci USA 111:1509–1514.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Joo HM,
    2. Kim JY,
    3. Jeong JB,
    4. Seong KM,
    5. Nam SY,
    6. Yang KH,
    7. Kim CS,
    8. Kim HS,
    9. Jeong M,
    10. An S, et al.
    (2011) Ret finger protein 2 enhances ionizing radiation-induced apoptosis via degradation of AKT and MDM2. Eur J Cell Biol 90:420–431.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kawai T and
    2. Akira S
    (2007) Signaling to NF-κB by Toll-like receptors. Trends Mol Med 13:460–469.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kristariyanto YA,
    2. Abdul Rehman SA,
    3. Campbell DG,
    4. Morrice NA,
    5. Johnson C,
    6. Toth R, and
    7. Kulathu Y
    (2015) K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of k29 polyubiquitin. Mol Cell 58:83–94.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Lerner M,
    2. Corcoran M,
    3. Cepeda D,
    4. Nielsen ML,
    5. Zubarev R,
    6. Pontén F,
    7. Uhlén M,
    8. Hober S,
    9. Grandér D, and
    10. Sangfelt O
    (2007) The RBCC gene RFP2 (Leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD. Mol Biol Cell 18:1670–1682.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Li Q,
    2. Yan J,
    3. Mao AP,
    4. Li C,
    5. Ran Y,
    6. Shu HB, and
    7. Wang YY
    (2011) Tripartite motif 8 (TRIM8) modulates TNFα- and IL-1β-triggered NF-κB activation by targeting TAK1 for K63-linked polyubiquitination. Proc Natl Acad Sci USA 108:19341–19346.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Li Y,
    2. Wu H,
    3. Wu W,
    4. Zhuo W,
    5. Liu W,
    6. Zhang Y,
    7. Cheng M,
    8. Chen YG,
    9. Gao N,
    10. Yu H, et al.
    (2014) Structural insights into the TRIM family of ubiquitin E3 ligases. Cell Res 24:762–765.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Liu J,
    2. Han C,
    3. Xie B,
    4. Wu Y,
    5. Liu S,
    6. Chen K,
    7. Xia M,
    8. Zhang Y,
    9. Song L,
    10. Li Z, et al.
    (2014) Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO. Nat Immunol 15:612–622.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Lyu JH,
    2. Park DW,
    3. Huang B,
    4. Kang SH,
    5. Lee SJ,
    6. Lee C,
    7. Bae YS,
    8. Lee JG, and
    9. Baek SH
    (2015) RGS2 suppresses breast cancer cell growth via a MCPIP1-dependent pathway. J Cell Biochem 116:260–267.
    OpenUrlCrossRefPubMed
  22. ↵
    1. McEwan WA,
    2. Tam JC,
    3. Watkinson RE,
    4. Bidgood SR,
    5. Mallery DL, and
    6. James LC
    (2013) Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nat Immunol 14:327–336.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Michel MA,
    2. Elliott PR,
    3. Swatek KN,
    4. Simicek M,
    5. Pruneda JN,
    6. Wagstaff JL,
    7. Freund SM, and
    8. Komander D
    (2015) Assembly and specific recognition of k29- and k33-linked polyubiquitin. Mol Cell 58:95–109.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Mullick AE,
    2. Tobias PS, and
    3. Curtiss LK
    (2005) Modulation of atherosclerosis in mice by Toll-like receptor 2. J Clin Invest 115:3149–3156.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Narayan K,
    2. Waggoner L,
    3. Pham ST,
    4. Hendricks GL,
    5. Waggoner SN,
    6. Conlon J,
    7. Wang JP,
    8. Fitzgerald KA, and
    9. Kang J
    (2014) TRIM13 is a negative regulator of MDA5-mediated type I interferon production. J Virol 88:10748–10757.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Niida M,
    2. Tanaka M, and
    3. Kamitani T
    (2010) Downregulation of active IKK beta by Ro52-mediated autophagy. Mol Immunol 47:2378–2387.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Noguchi K,
    2. Okumura F,
    3. Takahashi N,
    4. Kataoka A,
    5. Kamiyama T,
    6. Todo S, and
    7. Hatakeyama S
    (2011) TRIM40 promotes neddylation of IKKγ and is downregulated in gastrointestinal cancers. Carcinogenesis 32:995–1004.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Park Y,
    2. Jin HS,
    3. Aki D,
    4. Lee J, and
    5. Liu YC
    (2014) The ubiquitin system in immune regulation. Adv Immunol 124:17–66.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Poole E,
    2. Groves I,
    3. MacDonald A,
    4. Pang Y,
    5. Alcami A, and
    6. Sinclair J
    (2009) Identification of TRIM23 as a cofactor involved in the regulation of NF-κB by human cytomegalovirus. J Virol 83:3581–3590.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Rajsbaum R,
    2. García-Sastre A, and
    3. Versteeg GA
    (2014) TRIMmunity: the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity. J Mol Biol 426:1265–1284.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Shi M,
    2. Deng W,
    3. Bi E,
    4. Mao K,
    5. Ji Y,
    6. Lin G,
    7. Wu X,
    8. Tao Z,
    9. Li Z,
    10. Cai X, et al.
    (2008) TRIM30 alpha negatively regulates TLR-mediated NF-kappa B activation by targeting TAB2 and TAB3 for degradation. Nat Immunol 9:369–377.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Tomar D and
    2. Singh R
    (2014) TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNF induced NF-κB activation. Cell Signal 26:2606–2613.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Tomar D,
    2. Singh R,
    3. Singh AK,
    4. Pandya CD, and
    5. Singh R
    (2012) TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells. Biochim Biophys Acta 1823:316–326.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Uchil PD,
    2. Hinz A,
    3. Siegel S,
    4. Coenen-Stass A,
    5. Pertel T,
    6. Luban J, and
    7. Mothes W
    (2013) TRIM protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity. J Virol 87:257–272.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Versteeg GA,
    2. Rajsbaum R,
    3. Sánchez-Aparicio MT,
    4. Maestre AM,
    5. Valdiviezo J,
    6. Shi M,
    7. Inn KS,
    8. Fernandez-Sesma A,
    9. Jung J, and
    10. García-Sastre A
    (2013) The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors. Immunity 38:384–398.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Yuan WC,
    2. Lee YR,
    3. Lin SY,
    4. Chang LY,
    5. Tan YP,
    6. Hung CC,
    7. Kuo JC,
    8. Liu CH,
    9. Lin MY,
    10. Xu M, et al.
    (2014) K33-linked polyubiquitination of coronin 7 by Cul3-KLHL20 ubiquitin E3 ligase regulates protein trafficking. Mol Cell 54:586–600.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Zha J,
    2. Han KJ,
    3. Xu LG,
    4. He W,
    5. Zhou Q,
    6. Chen D,
    7. Zhai Z, and
    8. Shu HB
    (2006) The Ret finger protein inhibits signaling mediated by the noncanonical and canonical IκB kinase family members. J Immunol 176:1072–1080.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Zhang X,
    2. Zhang J,
    3. Zhang L,
    4. van Dam H, and
    5. ten Dijke P
    (2013) UBE2O negatively regulates TRAF6-mediated NF-κB activation by inhibiting TRAF6 polyubiquitination. Cell Res 23:366–377.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Zhao W,
    2. Wang L,
    3. Zhang M,
    4. Yuan C, and
    5. Gao C
    (2012) E3 ubiquitin ligase tripartite motif 38 negatively regulates TLR-mediated immune responses by proteasomal degradation of TNF receptor-associated factor 6 in macrophages. J Immunol 188:2567–2574.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Zhou F,
    2. Zhang X,
    3. van Dam H,
    4. Ten Dijke P,
    5. Huang H, and
    6. Zhang L
    (2012) Ubiquitin-specific protease 4 mitigates Toll-like/interleukin-1 receptor signaling and regulates innate immune activation. J Biol Chem 287:11002–11010.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools
Research ArticleArticle

Trim13 Stimulates NF-κB Activity via TRAF6 Ubiquitination

Bin Huang and Suk-Hwan Baek
Molecular Pharmacology April 1, 2017, 91 (4) 307-316; DOI: https://doi.org/10.1124/mol.116.106716
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section