The proinflammatory cytokine IL-1β is a crucial mediator of inflammatory responses. IL-1β–induced signaling is finely regulated by various mechanisms, and its imbalance is involved in a variety of diseases. In this study, we identified FAM177A1, a protein of unknown function, as a negative regulator of IL-1β–induced signaling in human cells. Overexpression of FAM177A1 inhibited IL-1β–triggered activation of NF-κB and transcription of inflammatory genes, whereas knockdown of FAM177A1 showed the opposite effects. Mechanistically, FAM177A1 competitively bound to the E3 ubiquitin ligase TRAF6 and impaired its interaction with the E2-conjugating enzyme Ubc13; therefore, it inhibited TRAF6-mediated polyubiquitination and recruitment of downstream signaling molecules. These findings reveal a function of FAM177A1 and promote our understanding of the regulatory mechanisms of IL-1β–induced inflammatory responses.

Inflammation is a host protective response against various stimuli, such as infection, tissue malfunction, and injury. The inflammatory response is coordinated by an extremely complicated network, which consists of numerous mediators, including cytokines, chemokines, and regulatory cells (1). As one of the most important proinflammatory cytokines, IL-1β belongs to the family of ILs, which affect a broad spectrum of immune and inflammatory responses (2). IL-1β–induced signaling is delicately regulated, and its imbalance is involved in a variety of diseases, including infectious diseases, autoimmune diseases, and cancers (3).

Upon IL-1β stimulation, IL-1 receptor (IL-1R) binds to the adaptor protein MyD88, which then recruits IL-1β–associated kinase 4 (IRAK4) and IRAK1/2 (4, 5). The E3 ubiquitin ligase TNFR-associated factor 6 (TRAF6) is subsequently recruited to the MyD88–IRAK4–IRAK1/2 complex and functions together with the E2-conjugating enzymes Ubc13/Uev1A to catalyze polyubiquitination, leading to the recruitment of TGF-β–activated kinase 1 (TAK1) complex and IκB kinase (IKK) complex through their ubiquitin-binding subunits TAK1-binding protein 2 (TAB2) and NEMO (NF-κB essential modulator), respectively. This leads to autophosphorylation and activation of TAK1, which further phosphorylates and activates IKKβ and MAPK kinases. Activated IKKβ and MAPK kinases activate transcription factors NF-κB and AP-1, resulting in induction of downstream inflammatory genes (610).

IL-1β–induced signaling is finely regulated to maintain a suitable level of inflammation and avoid excessive responses. Multiple mechanisms have been reported to participate in this process. For example, MARCH3 catalyzes K48-linked ubiquitination of IL-1R, leading to its degradation and attenuation of IL-1β–induced signaling (11). TRIM38 promotes TAB2/3 degradation via the lysosomal pathway to inhibit IL-1β–triggered inflammatory responses (12). A number of deubiquitinating enzymes, such as A20, USP4, and USP19, have been found to impair TRAF6-induced polyubiquitination and function as negative regulators of IL-1β signaling (1315). However, whether additional regulatory mechanisms exist is of great interest to explore.

Family with sequence similarity 177 member A1 (FAM177A1) is a protein universally expressed with unknown function. In this study, we identified FAM177A1 as a negative regulator of IL-1β–induced signaling. FAM177A1 inhibited TRAF6-mediated polyubiquitination and recruitment of downstream signaling molecules by disrupting the interaction between TRAF6 and the E2-conjugating enzyme Ubc13. Our study revealed the function of FAM177A1 and shed new light on the regulatory mechanisms of IL-1β–induced signaling.

Recombinant human IL-1β (PeproTech), LPS (Sigma-Aldrich), a dual-specific luciferase assay kit (Promega), RNase inhibitor (Thermo Fisher Scientific), SYBR Green (Bio-Rad Laboratories), and polybrene (MilliporeSigma) were purchased from the indicated manufacturers.

Abs against Flag (Sigma-Aldrich), HA (BioLegend), His (AE003; ABclonal), β-actin (AC004; ABclonal,), Myc-tag (9B11; Cell Signaling Technology), p-IKKα (Ser176)/IKKβ (Ser177, C84E11; Cell Signaling Technology), IKKβ (D30C6; Cell Signaling Technology), p-p38 (Thr180/Tyr182, 28B10; Cell Signaling Technology), p38 (Cell Signaling Technology), IRAK1 (D51G7; Cell Signaling Technology), p-TAK1 (Thr187; Cell Signaling Technology), TAK1 (D94D7; Cell Signaling Technology), p-p65 (Ser536, 93H1; Cell Signaling Technology), TRAF6 (D21G3; Cell Signaling Technology), TAB2 (C88H10; Cell Signaling Technology), p65 (C-20; Santa Cruz Biotechnology), ubiquitin (Santa Cruz Biotechnology), FAM177A1 (Novus Biologicals), and β-tubulin (Earthox Life Sciences) were purchased from the indicated manufacturers.

Human embryonic kidney cells (HEK293T) (CRL-11268), human colorectal carcinoma cells (HCT116) (CCL-247), and human peripheral blood monocytes (THP-1) (TIB-202) were purchased from American Type Culture Collection.

Expression plasmids of His-FAM177A1, FAM177A1-3xFlag, FAM177A1-HA, GST-TRAF6, Flag-TRAF6, HA-TRAF6, His-Ubc13, Flag-Ubc13, Flag-Ubc5c, and their mutants were constructed by standard molecular biology techniques. Other plasmids used in this study were previously described (12, 15, 16).

HEK293T cells were transfected by a standard calcium phosphate precipitation protocol. Twenty-four hours after transfection, cells were treated with the indicated stimuli or left untreated for the indicated time and then were harvested for luciferase assays (Promega).

HEK293T cells were transfected with two packaging plasmids (pGAG-Pol and pVSV-G [vesicular stomatitis virus G protein plasmid]) and empty vector or indicated plasmids, respectively, by calcium phosphate precipitation. Twelve hours later, media were replaced with antibiotic-free media. Sixty hours after transfection, the recombinant virus–containing media were harvested and filtered with a 0.22-μm filter and added to THP-1 or HCT116 cells in the presence of polybrene (8 μg/ml). Infected cells were selected with puromycin (1 μg/ml) for at least two passages before experiments.

Total RNAs were isolated from cells by using TRIzol reagent and reverse transcribed by using HiScript II Q RT SuperMix (Vazyme Biotech). Then cDNAs were subjected to qPCR to measure the mRNA expression level of the indicated genes, which were normalized to GAPDH. The sequences of specific primers for qPCR were as follows: GAPDH, forward: 5′-GACAAGCTTCCCGTTCTCAG-3′, reverse: 5′-GAGTCAACGGATTTGGTGGT-3′; IL8, forward: 5′-GAGAGTGATTGAGAGTGGACCAC-3′, reverse: 5′-CACAACCCTCTGCACCCAGTTT-3′; CXCL1, forward: 5′-CTTCAGGAACAGCCACCAGT-3′, reverse 5′-TCCTGCATCCCCCATAGTTA-3′; CXCL2, forward: 5′-CAAGAACATCCAAAGTGTGA-3′, reverse: 5′-CCATTCTTGAGTGTGGCTAT-3′; FAM177A1, forward: 5′-TGTGGAACGAGGAGAAGCCGTC-3′, reverse 5′-CCAGTTCTACATTTTCAAAGCCTC-3′; IFNB1, forward: 5′-TTGTTGAGAACCTCCTGGCT-3′, reverse: 5′-TGACTATGGTCCAGGCACAG-3′; ISG56, forward: 5′-TCATCAGGTCAAGGATAGTC-3′, reverse: 5′-CCACACTGTATTTGGTGTCTACG-3′; TNFA, forward: 5′-GCCGCATCGCCGTCTCCTAC-3′, reverse: 5′-CCTCAGCCCCCTCTGGGGTC-3′.

Cells were lysed in Nonidet P-40 lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) with protease and phosphatase inhibitors. For each immunoprecipitation, lysate was incubated with IgG or the indicated Ab (0.5 μg) and 15 μl of Protein G Sepharose (GE Healthcare) at 4°C for 3 h or overnight. The Protein G Sepharose beads were washed three times with 1 ml of lysis buffer containing 0.5 M NaCl. A Western blot assay was performed according to standard procedures.

Double-stranded oligonucleotides corresponding to the targeting sequences were cloned into the pSuper-Retro-RNAi plasmid (OligoEngine). The following sequences were targeted for human FAM177A1 RNAi: no. 1: 5′-GGGCAGATGAGTAACGAAA-3′; no. 2: 5′-GAAGAAAGTCCCAAGGAGA-3′.

GST pull-down assays were performed similarly to a previously described method (17). In brief, HEK293T cells were transfected with Flag-Ubc13 (5 μg), and, 24 h later, cells were harvested and lysed. Then, cell lysates and purified recombinant protein (0, 5, or 20 μg of His-FAM177A1) were incubated together with GST or GST-TRAF6 and Glutathione Sepharose (GE Healthcare) for 2 h at 4°C. Then beads were washed three times and subjected to Western blot assays.

An unpaired two-tailed Student t test was used for statistical analysis in Microsoft Excel, and p < 0.05 was considered significant.

To identify additional proteins that regulate IL-1β–induced signaling, we screened ∼16,000 independent human cDNA expression plasmids by luciferase reporter assays and found that FAM177A1 negatively regulated IL-1β–triggered NF-κB activation (Supplemental Fig. 1A). FAM177A1 is widely expressed in multiple primary tissues, such as brain, colon, liver, and lung (Supplemental Fig. 1B). There are two isoforms of FAM177A1 that consist of 213 (isoform 1) and 236 (isoform 2) aa, respectively, in humans. The two isoforms are nearly identical, except that the longer isoform possesses a signal peptide. Further investigation found that both isoforms were expressed in various cell lines, including human foreskin fibroblasts and HeLa, A549, HCT116, and THP-1 cells (Supplemental Fig. 1C). We then constructed expression plasmids of both FAM177A1 isoforms to confirm their functions. As shown in (Fig. 1A and 1B, overexpression of either isoform of FAM177A1 in HEK293T cells inhibited IL-1β–induced NF-κB activation in a dose-dependent manner, but it had no effects on IFN-γ–triggered activation of the IFN regulatory factor 1 (IRF1) promoter. Furthermore, we established HCT116 cells stably expressing either isoform of FAM177A1 and found that overexpression of either isoform of FAM177A1 inhibited IL-1β–induced transcription of proinflammatory cytokines, including CXCL1, CXCL2, and IL8 (Fig. 1C and 1D). Consistently, both isoforms of FAM177A1 suppressed IL-1β–triggered phosphorylation of TAK1, IKKα/β, p65, and p38, which are hallmarks of their activation (Fig. 1F). In contrast, IFN-γ–induced transcription of IRF1 was comparable between control and FAM177A1-expressing cells (Fig. 1E). These data suggest that overexpression of FAM177A1 inhibits IL-1β–induced signaling.

FIGURE 1.

Overexpression of FAM177A1 inhibits IL-1β–induced signaling. (A) Effects of FAM177A1 on IL-1β–induced NF-κB activation. HEK293T cells were transfected with pNifty-Luciferase reporter (1 ng; InvivoGen), PRL-TK (Renilla luciferase) reporter plasmid (10 ng), and FAM177A1 plasmid (50, 100, and 200 ng). Twenty-four hours after transfection, cells were treated with IL-1β (10 ng/ml) or left untreated for 12 h before reporter assays. The pNifty-Luciferase reporter plasmid contains five NF-κB binding sites. (B) Effects of FAM177A1 on IFN-γ–triggered activation of the IRF1 promoter. The experiments were performed similarly to those in (A), except that the IRF1 promoter reporter was used (100 ng) and cells were treated with IFN-γ (100 ng/ml). (C) Expression of FAM177A1 in stable cell lines. FAM177A1-overexpressed HCT116 stable cell lines were subjected to Western blot assays with the indicated Abs. (D) Effects of FAM177A1 on IL-1β–induced transcription of cytokines. FAM177A1-overexpressed HCT116 stable cell lines were treated with IL-1β (10 ng/ml) or left untreated for 2 h before RT-qPCR analysis. (E) Effects of FAM177A1 on IL-1β–induced phosphorylation of TAK1, IKKα/β, p65, and p38. FAM177A1-overexpressed HCT116 stable cell lines were treated with IL-1β (10 ng/ml) or left untreated for the indicated times before Western blot assays. (F) Effects of FAM177A1 on IFN-γ–induced transcription of IRF1. FAM177A1-overexpressed HCT116 stable cell lines were treated with IFN-γ (100 ng/ml) or left untreated for 6 h before RT-qPCR analysis. (A and D) Graphs show mean ± SD. *p < 0.05.

FIGURE 1.

Overexpression of FAM177A1 inhibits IL-1β–induced signaling. (A) Effects of FAM177A1 on IL-1β–induced NF-κB activation. HEK293T cells were transfected with pNifty-Luciferase reporter (1 ng; InvivoGen), PRL-TK (Renilla luciferase) reporter plasmid (10 ng), and FAM177A1 plasmid (50, 100, and 200 ng). Twenty-four hours after transfection, cells were treated with IL-1β (10 ng/ml) or left untreated for 12 h before reporter assays. The pNifty-Luciferase reporter plasmid contains five NF-κB binding sites. (B) Effects of FAM177A1 on IFN-γ–triggered activation of the IRF1 promoter. The experiments were performed similarly to those in (A), except that the IRF1 promoter reporter was used (100 ng) and cells were treated with IFN-γ (100 ng/ml). (C) Expression of FAM177A1 in stable cell lines. FAM177A1-overexpressed HCT116 stable cell lines were subjected to Western blot assays with the indicated Abs. (D) Effects of FAM177A1 on IL-1β–induced transcription of cytokines. FAM177A1-overexpressed HCT116 stable cell lines were treated with IL-1β (10 ng/ml) or left untreated for 2 h before RT-qPCR analysis. (E) Effects of FAM177A1 on IL-1β–induced phosphorylation of TAK1, IKKα/β, p65, and p38. FAM177A1-overexpressed HCT116 stable cell lines were treated with IL-1β (10 ng/ml) or left untreated for the indicated times before Western blot assays. (F) Effects of FAM177A1 on IFN-γ–induced transcription of IRF1. FAM177A1-overexpressed HCT116 stable cell lines were treated with IFN-γ (100 ng/ml) or left untreated for 6 h before RT-qPCR analysis. (A and D) Graphs show mean ± SD. *p < 0.05.

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To examine whether endogenous FAM177A1 negatively regulates IL-1β–induced signaling, we constructed two FAM177A1 RNAi plasmids and confirmed their inhibition of the expression of both isoforms of FAM177A1 (Fig. 2A). Knockdown of FAM177A1 enhanced IL-1β–triggered NF-κB activation and transcription of inflammatory genes such as CXCL1, CXCL2, and IL8 (Fig. 2B and 2C). Consistently, IL-1β–induced phosphorylation of TAK1, IKKα/β, p65, and p38, as well as degradation of IκBα, were also promoted in FAM177A1-knockdown cells (Fig. 2D). In contrast, knockdown of FAM177A1 had little effect on IFN-γ–induced activation of IRF1 or transcription of the IRF1 gene (Fig. 2B and 2E).

FIGURE 2.

Knockdown of FAM177A1 enhances IL-1β–induced signaling. (A) Efficiency of FAM177A1 RNAi on FAM177A1 expression in HCT116 stable knockdown cell lines. Cell lysates were subjected to Western blot assays with the indicated Abs. (B) Effects of FAM177A1 knockdown on IL-1β–induced NF-κB activation and IFN-γ–triggered activation of the IRF1 promoter. HCT116 stable knockdown cell lines were transfected with pNifty-Luciferase reporter (1 ng) or IRF1 promoter reporter (100 ng) and PRL-TK (Renilla luciferase) reporter plasmid (10 ng). Twenty-four hours after transfection, cells were treated with IL-1β (10 ng/ml) or IFN-γ (100 ng/ml) or left untreated for 12 h before reporter assays. (C) Effects of FAM177A1 knockdown on IL-1β–induced transcription of cytokines. HCT116 stable knockdown cell lines were treated with IL-1β (10 ng/ml) or left untreated for 1, 2, or 3 h before RT-qPCR analysis. (D) Effects of FAM177A1 knockdown on IL-1β–induced phosphorylation of TAK1, IKKα/β, p65, p38, and degradation of IκBα. HCT116 stable knockdown cell lines were treated with IL-1β (10 ng/ml) or left untreated for the indicated times before Western blot assays. (E) Effects of FAM177A1 knockdown on IFN-γ–triggered transcription of the IRF1 gene. The experiments were performed similarly to those in (C), except that cells were treated with IFN-γ (100 ng/ml). (F) Effects of FAM177A1 knockdown on LPS-induced transcription of inflammatory genes. THP-1 stable knockdown cell lines were treated with LPS or left untreated for 2 h before RT-qPCR. (B, C, and F) Graphs show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Knockdown of FAM177A1 enhances IL-1β–induced signaling. (A) Efficiency of FAM177A1 RNAi on FAM177A1 expression in HCT116 stable knockdown cell lines. Cell lysates were subjected to Western blot assays with the indicated Abs. (B) Effects of FAM177A1 knockdown on IL-1β–induced NF-κB activation and IFN-γ–triggered activation of the IRF1 promoter. HCT116 stable knockdown cell lines were transfected with pNifty-Luciferase reporter (1 ng) or IRF1 promoter reporter (100 ng) and PRL-TK (Renilla luciferase) reporter plasmid (10 ng). Twenty-four hours after transfection, cells were treated with IL-1β (10 ng/ml) or IFN-γ (100 ng/ml) or left untreated for 12 h before reporter assays. (C) Effects of FAM177A1 knockdown on IL-1β–induced transcription of cytokines. HCT116 stable knockdown cell lines were treated with IL-1β (10 ng/ml) or left untreated for 1, 2, or 3 h before RT-qPCR analysis. (D) Effects of FAM177A1 knockdown on IL-1β–induced phosphorylation of TAK1, IKKα/β, p65, p38, and degradation of IκBα. HCT116 stable knockdown cell lines were treated with IL-1β (10 ng/ml) or left untreated for the indicated times before Western blot assays. (E) Effects of FAM177A1 knockdown on IFN-γ–triggered transcription of the IRF1 gene. The experiments were performed similarly to those in (C), except that cells were treated with IFN-γ (100 ng/ml). (F) Effects of FAM177A1 knockdown on LPS-induced transcription of inflammatory genes. THP-1 stable knockdown cell lines were treated with LPS or left untreated for 2 h before RT-qPCR. (B, C, and F) Graphs show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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It has been demonstrated that IL-1β and LPS activate a similar signaling cascade that is mediated by the MyD88–TRAF6 axis. We generated FAM177A1-knockdown THP-1 cells to examine whether FAM177A1 also regulates IL-1β–/LPS-induced signaling in monocytes. As shown in (Fig. 2F, LPS-triggered transcription of CXCL1, CXCL2, and IL8 genes was markedly enhanced in FAM177A1-knockdown THP-1 cells. Because the expression of IL-1R1 is low in THP-1 cells, we used PMA to differentiate THP-1 cells into macrophage-like cells, which express higher levels of IL-1R1, and then investigated the function of FAM177A1 on IL-1β–induced signaling. As shown in Supplemental Fig. 2A, knockdown of FAM177A1 enhanced IL-1β–triggered transcription of inflammatory genes in PMA-differentiated THP-1 cells (Supplemental Fig. 2A). These results suggest that endogenous FAM177A1 negatively regulates IL-1β–/LPS-induced signaling.

We next investigated the molecular mechanisms by which FAM177A1 regulates IL-1β–induced signaling. Reporter assays showed that knockdown of FAM177A1 potentiated NF-κB activation mediated by MyD88 and TRAF6 but not the downstream molecules such as TAK1/TAB1, IKKβ, or p65 (Fig. 3A), indicating that it targets a step at the level of or upstream of TRAF6. In transient transfection and coimmunoprecipitation experiments, FAM177A1 interacted with IRAK1-KD (the kinase-dead mutant that is often used for coimmunoprecipitation), TRAF6, and TAK1 (Fig. 3B). Endogenous coimmunoprecipitation experiments found that FAM177A1 interacted with TRAF6, and their interaction was enhanced after IL-1β stimulation (Fig. 3C). In the same experiments, endogenous FAM177A1 also associated with polyubiquitinated IRAK1 and TAK1 (Fig. 3C), but whether such association is direct or indirect is unknown. In vitro GST pull-down assays further indicated that FAM177A1 directly interacted with TRAF6 (Fig. 3D). Taken together, these findings indicate that FAM177A1 may target TRAF6 to inhibit IL-1β–induced signaling.

FIGURE 3.

FAM177A1 regulates IL-1β–induced signaling at TRAF6 level. (A) Knockdown of FAM177A1 potentiated NF-κB activation mediated by MyD88 and TRAF6 but not downstream molecules, including TAK1 + TAB1, IKKβ, or p65. The HCT116 control cell line and FAM177A1 stable knockdown cell line were transfected with pNifty-Luciferase reporter (1 ng), PRL-TK (Renilla luciferase) reporter plasmid (10 ng), and empty vector or the indicated plasmids expressing MyD88,TRAF6, TAK1, TAB1, IKKβ, and p65 (50 ng). Twenty-four hours after transfection, cells were harvested for reporter assays. (B) FAM177A1 interacts with TRAF6. HEK293T cells were transfected with plasmid expressing HA-tagged FAM177A1 and the indicated plasmids expressing Flag-tagged MyD88, TRAF6, IRAK4KD, IRAK1KD, TAK1, TAB2, IKKα, IKKβ, and p65. Twenty-four hours later, transfected cells were harvested and lysed, then subjected to coimmunoprecipitation experiments with IgG or anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blot (IB) analysis with anti-HA or anti-Flag. (C) Endogenous interaction between FAM177A1 and TRAF6. HCT116 cells were treated with IL-1β (50 ng/ml) or left untreated for the indicated times and then were harvested and lysed. The cell lysates were then subjected to coimmunoprecipitation experiments with IgG or anti-FAM177A1. The immunoprecipitation samples and the rest of the cell lysates were subjected to Western blot analysis with the indicated Abs. (D) TRAF6 interacts with FAM177A1 directly. Prokaryotically expressed GST or GST-TRAF6 and His-FAM177A1 were subjected to in vitro GST pull-down assay. The pull-down samples and input samples were then subjected to Coomassie Brilliant Blue (CBB) staining or Western blotting with anti-His. (A) Graphs show mean ± SD. *p < 0.05, **p < 0.01.

FIGURE 3.

FAM177A1 regulates IL-1β–induced signaling at TRAF6 level. (A) Knockdown of FAM177A1 potentiated NF-κB activation mediated by MyD88 and TRAF6 but not downstream molecules, including TAK1 + TAB1, IKKβ, or p65. The HCT116 control cell line and FAM177A1 stable knockdown cell line were transfected with pNifty-Luciferase reporter (1 ng), PRL-TK (Renilla luciferase) reporter plasmid (10 ng), and empty vector or the indicated plasmids expressing MyD88,TRAF6, TAK1, TAB1, IKKβ, and p65 (50 ng). Twenty-four hours after transfection, cells were harvested for reporter assays. (B) FAM177A1 interacts with TRAF6. HEK293T cells were transfected with plasmid expressing HA-tagged FAM177A1 and the indicated plasmids expressing Flag-tagged MyD88, TRAF6, IRAK4KD, IRAK1KD, TAK1, TAB2, IKKα, IKKβ, and p65. Twenty-four hours later, transfected cells were harvested and lysed, then subjected to coimmunoprecipitation experiments with IgG or anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blot (IB) analysis with anti-HA or anti-Flag. (C) Endogenous interaction between FAM177A1 and TRAF6. HCT116 cells were treated with IL-1β (50 ng/ml) or left untreated for the indicated times and then were harvested and lysed. The cell lysates were then subjected to coimmunoprecipitation experiments with IgG or anti-FAM177A1. The immunoprecipitation samples and the rest of the cell lysates were subjected to Western blot analysis with the indicated Abs. (D) TRAF6 interacts with FAM177A1 directly. Prokaryotically expressed GST or GST-TRAF6 and His-FAM177A1 were subjected to in vitro GST pull-down assay. The pull-down samples and input samples were then subjected to Coomassie Brilliant Blue (CBB) staining or Western blotting with anti-His. (A) Graphs show mean ± SD. *p < 0.05, **p < 0.01.

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Previous studies have shown that the different domains of TRAF6 play distinct roles in IL-1β–induced signaling (18, 19). Domain-mapping experiments found that the coiled-coil (CC) domain of TRAF6 is important for its interaction with FAM177A1 (Fig. 4A). Furthermore, FAM177A1 only inhibited NF-κB activation induced by TRAF6 truncations containing the CC domain (Fig. 4B), suggesting that this domain is required for FAM177A1-mediated inhibition of TRAF6. It has been reported that the CC domain of TRAF6 is responsible for its polymerization as well as its association with the E2-conjugating enzyme Ubc13. Therefore, we tested whether FAM177A1 affected these two processes. Interestingly, FAM177A1 inhibited the association between TRAF6 and Ubc13 in a dose-dependent manner but had no effects on the self-association of TRAF6 (Fig. 4C and 4D). Moreover, in vitro GST pull-down assays showed that the direct interaction between TRAF6 and Ubc13 was impaired by FAM177A1 in a dose-dependent manner (Fig. 4E), indicating that FAM177A1 competes with Ubc13 for TRAF6.

FIGURE 4.

FAM177A1 inhibits TRAF6–Ubc13 interaction. (A) The interaction between FAM177A1 and truncation mutants of TRAF6. A schematic diagram of truncation mutants of TRAF6 is shown (upper panel). HEK293T cells were transfected with the indicated plasmids expressing Flag-tagged FAM177A1 and HA-tagged truncation mutants of TRAF6. Twenty-four hours later, transfected cells were harvested to perform coimmunoprecipitation experiments with IgG or anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blotting (IB) with anti-HA or anti-Flag. (B) Effects of FAM177A1 on NF-κB activation mediated by truncation mutants of TRAF6. HEK293T cells were transfected with pNifty-Luciferase reporter (1 ng) and the indicated plasmids expressing truncation mutants of TRAF6 (50 ng) and FAM177A1 or empty vector (200 ng). Twenty-four hours after transfection, cells were subjected to reporter assays. (C) Effects of FAM177A1 on TRAF6 oligomerization. HEK293T cells were transfected with the indicated plasmids expressing Flag-tagged Ubc13 and HA-tagged TRAF6 and empty vector or a different dose of plasmid expressing tag-free FAM177A1. Twenty-four hours later, transfected cells were harvested to perform coimmunoprecipitation experiments with anti-Flag. The cell lysates and immunoprecipitation samples were then subjected to Western blot analysis with anti-HA or anti-Flag. (D) Effects of FAM177A1 on TRAF6–Ubc13 interaction. The experiments were performed similarly to those in (C). (E) The direct interaction between FAM177A1 and TRAF6 impaired TRAF6–Ubc13 interaction in vitro. HEK293T cells were transfected with Flag-tagged Ubc13. Twenty-four hours later, cells were lysed and subjected to in vitro pull-down assay with prokaryotically expressed GST or GST-TRAF6 and a different dose of His-FAM177A1. The pull-down samples and input samples were then subjected to Coomassie Brilliant Blue (CBB) staining or Western blotting with anti-His or anti-Flag. (F) Effects of FAM177A1 on TRAF6–Ubc5c interaction. The experiments were performed similarly to those in (C). (G) Effects of FAM177A1 knockdown on Sendai virus (SeV)-induced signaling. HCT116 stable knockdown cell lines were treated with SeV or left untreated for 12 h before RT-qPCR. (B and G) Graphs show mean ± SD. *p < 0.05, **p < 0.01.

FIGURE 4.

FAM177A1 inhibits TRAF6–Ubc13 interaction. (A) The interaction between FAM177A1 and truncation mutants of TRAF6. A schematic diagram of truncation mutants of TRAF6 is shown (upper panel). HEK293T cells were transfected with the indicated plasmids expressing Flag-tagged FAM177A1 and HA-tagged truncation mutants of TRAF6. Twenty-four hours later, transfected cells were harvested to perform coimmunoprecipitation experiments with IgG or anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blotting (IB) with anti-HA or anti-Flag. (B) Effects of FAM177A1 on NF-κB activation mediated by truncation mutants of TRAF6. HEK293T cells were transfected with pNifty-Luciferase reporter (1 ng) and the indicated plasmids expressing truncation mutants of TRAF6 (50 ng) and FAM177A1 or empty vector (200 ng). Twenty-four hours after transfection, cells were subjected to reporter assays. (C) Effects of FAM177A1 on TRAF6 oligomerization. HEK293T cells were transfected with the indicated plasmids expressing Flag-tagged Ubc13 and HA-tagged TRAF6 and empty vector or a different dose of plasmid expressing tag-free FAM177A1. Twenty-four hours later, transfected cells were harvested to perform coimmunoprecipitation experiments with anti-Flag. The cell lysates and immunoprecipitation samples were then subjected to Western blot analysis with anti-HA or anti-Flag. (D) Effects of FAM177A1 on TRAF6–Ubc13 interaction. The experiments were performed similarly to those in (C). (E) The direct interaction between FAM177A1 and TRAF6 impaired TRAF6–Ubc13 interaction in vitro. HEK293T cells were transfected with Flag-tagged Ubc13. Twenty-four hours later, cells were lysed and subjected to in vitro pull-down assay with prokaryotically expressed GST or GST-TRAF6 and a different dose of His-FAM177A1. The pull-down samples and input samples were then subjected to Coomassie Brilliant Blue (CBB) staining or Western blotting with anti-His or anti-Flag. (F) Effects of FAM177A1 on TRAF6–Ubc5c interaction. The experiments were performed similarly to those in (C). (G) Effects of FAM177A1 knockdown on Sendai virus (SeV)-induced signaling. HCT116 stable knockdown cell lines were treated with SeV or left untreated for 12 h before RT-qPCR. (B and G) Graphs show mean ± SD. *p < 0.05, **p < 0.01.

Close modal

It has been well established that TRAF6 pairs with distinct E2-conjugating enzymes when it participates in different signaling pathways (10, 20). For example, in RIG-I–mediated signaling, which is pivotal for host recognition and response to viral RNA, TRAF6 partners with Ubc5 but not Ubc13 to activate the signaling (21, 22). We then determined whether FAM177A1 also affects the interaction of TRAF6 with other E2s, such as Ubc5. As shown in (Fig. 4F, overexpression of FAM177A1 had no effects on TRAF6–Ubc5c interaction. Consistently, knockdown of FAM177A1 had little effect on Sendai virus–induced transcription of downstream antiviral genes (Fig. 4G). These results suggest that FAM177A1 specifically disrupts the interaction between TRAF6 and Ubc13.

It has previously been reported that aa 314–320 in the CC domain of mouse TRAF6 (mTRAF6) are essential for its interaction with Ubc13 (18). Alignment of this region between mouse and human TRAF6 revealed its conservation. We then examined whether FAM177A1 targets these residues to block TRAF6–Ubc13 interaction. First, we generated a truncation mutant of human TRAF6 that lacks aa 305–311 (hTRAF6Δ305–311) and tested its interaction with Ubc13. Similarly to mTRAF6, hTRAF6Δ305–311 obviously lost its ability to interact with Ubc13 (Fig. 5A). In similar experiments, we noticed that hTRAF6Δ305–311 did not affect TRAF6 oligomerization, which is a prerequisite for its interaction with Ubc13 (Fig. 5B). We next determined whether FAM177A1 targets this region to inhibit TRAF6–Ubc13 interaction. As shown in (Fig. 5C, loss of aa 305–311 significantly dampened the association of TRAF6 with FAM177A1, suggesting that FAM177A1 also binds to this region of TRAF6. Taken together, these findings indicate that FAM177A1 and Ubc13 bind to the same region of TRAF6; therefore, FAM177A1 competitively inhibits TRAF6–Ubc13 interaction.

FIGURE 5.

FAM177A1 competes with Ubc13 to bind to the key region in the CC domain of TRAF6. (A) Effects of 305–311-aa deletion in the CC domain of TRAF6 on its association with Ubc13. Schematic diagram of 305–311-aa deletion is shown (upper panel). HEK293T cells were transfected with Flag-tagged Ubc13 (5 μg) and HA-tagged indicated mutant or full-length TRAF6 (3 μg). Twenty-four hours later, transfected cells were harvested to perform coimmunoprecipitation experiments with IgG or anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blotting with anti-HA or anti-Flag. (B) Effects of 305–311-aa deletion in the CC domain of TRAF6 on its oligomerization. The experiments were performed similarly to those in (A). (C) Effects of 305–311-aa deletion in the CC domain of TRAF6 on its association with FAM177A1. The experiments were performed similarly to those in (A).

FIGURE 5.

FAM177A1 competes with Ubc13 to bind to the key region in the CC domain of TRAF6. (A) Effects of 305–311-aa deletion in the CC domain of TRAF6 on its association with Ubc13. Schematic diagram of 305–311-aa deletion is shown (upper panel). HEK293T cells were transfected with Flag-tagged Ubc13 (5 μg) and HA-tagged indicated mutant or full-length TRAF6 (3 μg). Twenty-four hours later, transfected cells were harvested to perform coimmunoprecipitation experiments with IgG or anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blotting with anti-HA or anti-Flag. (B) Effects of 305–311-aa deletion in the CC domain of TRAF6 on its oligomerization. The experiments were performed similarly to those in (A). (C) Effects of 305–311-aa deletion in the CC domain of TRAF6 on its association with FAM177A1. The experiments were performed similarly to those in (A).

Close modal

It has been reported that TRAF6-mediated polyubiquitination in response to IL-1β is Ubc13 dependent (23). Multiple proteins, including TRAF6, IRAK1, and TAK1, could be modified by TRAF6-mediated polyubiquitination after IL-1β stimulation (2426). Polyubiquitin chains serve as a platform to recruit signaling components via ubiquitin-binding subunits (9, 10, 19, 23, 24, 26). For example, TAB2, a subunit of the TAK1 complex, binds to polyubiquitin chains and thus is recruited to the TRAF6–IRAK complex (8, 27). We then investigated the effects of FAM177A1 on TRAF6-mediated polyubiquitination and recruitment of signaling molecules. Overexpression of FAM177A1 inhibited autopolyubiquitination of TRAF6 (Fig. 6A). Endogenous coimmunoprecipitation showed that after IL-1β stimulation, polyubiquitination of TRAF6 and IRAK1 was increased in FAM177A1-knockdown cells (Fig. 6B). Furthermore, after IL-1β stimulation, TAB2-associated polyubiquitinated IRAK1 and polyubiquitinated TAK1 were elevated in FAM177A1-knockdown cells (Fig. 6C). Taken together, these results indicated that FAM177A1 negatively regulates IL-1β–induced, TRAF6-mediated polyubiquitination and recruitment of downstream molecules.

FIGURE 6.

FAM177A1 inhibits TRAF6-induced polyubiquitination. (A) Overexpression of FAM177A1 inhibited autopolyubiquitination of TRAF6. HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF6, Myc-tagged ubiquitin, and HA-tagged FAM177A1 or empty vector. Twenty-four hours after transfection, cells were harvested and denatured (95°C for 10 min in 1% SDS) before performing coimmunoprecipitation experiments with anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blot (IB) analysis with anti-Myc, anti-Flag, or anti-HA. (B) Knockdown of FAM177A1 enhanced TRAF6-mediated polyubiquitination upon IL-1β stimulation. The HCT116 control cell line and FAM177A1 stable knockdown cell line were treated with IL-1β (50 ng/ml) or left untreated for the indicated times. The cell lysates were then subjected to coimmunoprecipitation experiments with IgG or anti-TRAF6. The cell lysates and immunoprecipitation samples were then subjected to Western blotting with the indicated Abs. (C) Knockdown of FAM177A1 enhanced polyubiquitin-mediated recruitment of signaling molecules. The experiments were performed similarly to those in (B).

FIGURE 6.

FAM177A1 inhibits TRAF6-induced polyubiquitination. (A) Overexpression of FAM177A1 inhibited autopolyubiquitination of TRAF6. HEK293T cells were transfected with plasmids expressing Flag-tagged TRAF6, Myc-tagged ubiquitin, and HA-tagged FAM177A1 or empty vector. Twenty-four hours after transfection, cells were harvested and denatured (95°C for 10 min in 1% SDS) before performing coimmunoprecipitation experiments with anti-Flag. The cell lysates and immunoprecipitation (IP) samples were then subjected to Western blot (IB) analysis with anti-Myc, anti-Flag, or anti-HA. (B) Knockdown of FAM177A1 enhanced TRAF6-mediated polyubiquitination upon IL-1β stimulation. The HCT116 control cell line and FAM177A1 stable knockdown cell line were treated with IL-1β (50 ng/ml) or left untreated for the indicated times. The cell lysates were then subjected to coimmunoprecipitation experiments with IgG or anti-TRAF6. The cell lysates and immunoprecipitation samples were then subjected to Western blotting with the indicated Abs. (C) Knockdown of FAM177A1 enhanced polyubiquitin-mediated recruitment of signaling molecules. The experiments were performed similarly to those in (B).

Close modal

It is well known that TRAF6-Ubc13–mediated polyubiquitination plays a crucial role in IL-1β–induced signaling (7, 9, 10, 23). In this study, we identified FAM177A1 as a negative regulator of IL-1β–induced signaling by inhibiting TRAF6–Ubc13 interaction.

FAM177A1 has two isoforms, both of which are universally expressed in all cell lines we tested. Overexpression of each isoform of FAM177A1 inhibited IL-1β–induced NF-κB activation and transcription of downstream inflammatory genes, whereas knockdown of FAM177A1 had the opposite effects. In our experiments, we were not able to significantly knock down the expression of Fam177a in murine cell lines by short hairpin RNA, small interfering RNA, or single-guide RNA strategies. This may be due to the existence of Fam177a2, a copy gene of Fam177a in mice. Therefore, it remains to be further investigated whether FAM177A1 plays a similar role in mice.

Several lines of evidence suggest that FAM177A1 targets TRAF6 for its inhibitory function. First, FAM177A1 inhibited MyD88- and TRAF6- but not downstream protein-mediated NF-κB activation, indicating it targets a step at or upstream of TRAF6. Second, FAM177A1 directly interacts with TRAF6 (Fig. 3D), and their endogenous interaction increased after IL-1β stimulation. Third, FAM177A1 inhibits association of TRAF6 with the E2 Ubc13 by competitively binding to the motif of TRAF6, which is responsible for TRAF6–Ubc13 interaction. As a result of impaired TRAF6–Ubc13 interaction, TRAF6-mediated polyubiquitination and recruitment of downstream signaling molecules is inhibited by FAM177A1.

To our knowledge, this study reported a new function of FAM177A1 and revealed a mechanism that inhibits IL-1β–induced signaling by specifically dampening TRAF6–Ubc13 (E3–E2) interaction. Previously, it has been reported that A20 could promote the degradation of E2s, including Ubc13 and Ubc5c, thus disrupting the E3–E2 complexes (28). FAM177A1 functions in a distinct way from A20: It disrupts the E3–E2 complex by competitive interaction instead of inducing degradation. In addition, it specifically targets the interaction of TRAF6 with Ubc13 but not other E2s.

Given the importance and specific function of FAM177A1 in TRAF6–Ubc13 interaction, it is a potential target for the treatment of inflammatory diseases that involve TRAF6-Ubc13. Recently, Brenke et al. reported a compound that specifically disrupted TRAF6–Ubc13 interaction, which impaired inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in mouse models (29). Therefore, whether FAM177A1 could be used as a therapeutic target is worth further investigation.

We thank members of the Wang Lab for technical help and discussions.

This work was supported by grants from the Strategic Priority Research Program funded by the Chinese Academy of Sciences (XDB29010302) and the National Key R&D Project of China (2020YFC0841000).

B.-W.L., Z.-S.X., and Y.-Y.W. conceived and designed the study. B.-W.L., H.-Y.Z., W.-T.D., and Z.-S.X. performed the experiments. B.-W.L., Z.-S.X., Y.-Y.W., and Y.R. analyzed the data. B.-W.L., Z.-S.X., and Y.-Y.W. wrote the manuscript. All of the authors discussed the results and commented on the manuscript.

The online version of this article contains supplemental material.

Abbreviations used in this article

CC

coiled coil

IKK

IκB kinase

IRAK

IL-1β–associated kinase

IRF

IFN regulatory factor

qPCR

quantitative PCR

RNAi

RNA interference

TAB

TAK1-binding protein

TAK1

TGF-β–activated kinase 1

TRAF

TNFR-associated factor

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The authors have no financial conflicts of interest.

Supplementary data