TRAF [TNF (tumour necrosis factor)-receptor-associated factor] 2 and 6 are essential adaptor proteins for the NF-κB (nuclear factor κB) signalling pathway, which play important roles in inflammation and immune response. Polyubiquitination of TRAF2 and TRAF6 is critical to their activities and functions in TNFα- and IL (interleukin)-1β-induced NF-κB activation. However, the regulation of TRAF2 and TRAF6 by deubiquitination remains incompletely understood. In the present study, we identified USP (ubiquitin-specific protease) 4 as a novel deubiquitinase targeting TRAF2 and TRAF6 for deubiquitination. We found that USP4 specifically interacts with TRAF2 and TRAF6, but not TRAF3. Moreover, USP4 associates with TRAF6 both in vitro and in vivo, independent of its deubiquitinase activity. The USP domain is responsible for USP4 to interact with TRAF6. Ectopic expression of USP4 inhibits the TRAF2- and TRAF6-stimulated NF-κB reporter gene and negatively regulates the TNFα-induced IκBα (inhibitor of NF-κBα) degradation and NF-κB activation. Knockdown of USP4 significantly increased TNFα-induced cytokine expression. Furthermore, we found that USP4 deubiquitinates both TRAF2 and TRAF6 in vivo and in vitro in a deubiquitinase activity-dependent manner. Importantly, the results of the present study showed that USP4 is a negative regulator of TNFα- and IL-1β-induced cancer cell migration. Taken together, the present study provides a novel insight into the regulation of the NF-κB signalling pathway and uncovers a previously unknown function of USP4 in cancer.

INTRODUCTION

Protein ubiquitination is an essential post-translational modification with critical roles in various biological functions, such as cell growth, apoptosis, DNA damage repair, immune responses and neuron degeneration [13]. It is a process of covalently attaching one or more ubiquitins to the lysine residues of the targeted proteins triggered by an enzymatic cascade [4]. Ubiquitin is a highly conserved polypeptide of 76 amino acids with seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63). Most protein ubiquitination can be divided into mono-ubiquitination, and Lys48-linked and Lys63-linked polyubiquitination based on the length and linkage of ubiquitin chains. Lys48-linked polyubiquitination, where ubiquitin is attached to another ubiquitin on its Lys48 residue, is thought to target proteins for 26S proteasome-dependent degradation. Lys63-linked polyubiquitination mainly plays non-proteolytic roles in protein trafficking, DNA damage repair and activation of signalling pathways [4,5]. Three distinct classes of enzymes including E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) are involved in the protein ubiquitination process. E3 ubiquitin ligases containing a substrate recognition motif compose a large protein family with over 600 members and are responsible for determining the substrate specificity [6,7].

Ubiquitination is a reversible process by a family of DUBs (deubiquitinases). The ubiquitin can be directly removed from substrates by DUBs [8]. Currently, nearly 100 DUBs have been identified. According to their enzyme features, DUBs can be divided into two families: a metalloprotease family and a cysteine protease family. The cysteine protease family can be further divided into four subclasses based on their ubiquitin protease domains: USPs (ubiquitin-specific proteases), UCHs (ubiquitin C-terminal hydrolases), OTUs (otubain proteases), and MJDs (Machado–Joseph disease proteases). The USPs compose the biggest subfamily with more than 50 members [8]. However, the function of most DUBs remains unknown.

Ubiquitination/deubiquitination plays a critical role in the activation of the NF-κB (nuclear factor κB) signalling pathway, which has multiple functions in regulating cell proliferation, apoptosis and immune responses [9]. For example, both Lys63-linked polyubiquitination of IKKγ [IκB (inhibitor of NF-κB) kinase γ] and Lys48-linked polyubiquitination of IκBα are important to NF-κB activation [10]. Polyubiquitination of TRAF {TNFR [TNF (tumour necrosis factor) receptor]-associated factor} 6 is critical to its activity towards downstream targets to mediate IL (interleukin)-1β-induced NF-κB activation [11]. TRAF2 possesses an S1P (sphingosine 1-phosphate)-dependent E3 ubiquitin ligase activity [12] and its polyubiquitination has also been shown to be critical to TNFα-induced NF-κB activation [13]. Deubiquitination of TRAF2 or TRAF6 by DUBs markedly inhibits cytokines, such as TNFα- and IL-1β-mediated NF-κB activation. For example, CYLD, A20 and USP20 have been reported to negatively regulate NF-κB signalling, at least partially, through deubiquitinating TRAF2 and/or TRAF6 [1417].

In the present study, we identified USP4 as a novel deubiquitinase for both TRAF2 and TRAF6. We found that USP4 interacts with and deubiquitinates TRAF2 and TRAF6, and consequently inhibits TNFα- and IL-1β-induced NF-κB activation. Importantly, we found that USP4 negatively regulates TNFα-mediated migration of lung cancer cells.

EXPERIMENTAL

Cell culture and transfection

HEK (human embryonic kidney)-293T cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% heat-inactivated FBS (fetal bovine serum) and cells from the human lung adenocarcinoma epithelial cell line A549 were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS. The cells were incubated at 37°C in 5% CO2. Transfections were performed using calcium phosphate/DNA co-precipitation (for HEK-293T cells) and Lipofectamine™ 2000 (for A549 cells) according to the manufacturer's instructions. siRNA (small interfering RNA) oligonucleotides were transfected using Lipofectamine™ 2000.

Plasmids and siRNA

USP4, cIAP (cellular inhibitor of apoptosis) and TRAF2 were amplified from HEK-293T cells by RT (reverse transcription)–PCR and cloned into pcDNA3.1 vectors with a HA (haemagglutinin), FLAG or GFP (green fluorescent protein) tag at the N-terminus. TNFR1 and TNFR2 were cloned into pcDNA3.1 with a FLAG tag at the C-terminus. The USP4-C311S expression construct was generated using site-directed mutagenesis. Deletion mutants of USP4 were subcloned into the pCDNA3.1 vector with an HA tag at the N-terminus by standard cloning methods. GST (glutathione transferase)–TRAF6 and GST–USP4 were cloned into pGEX-4T-2. FLAG–TRAF6 and its deletion mutants were kindly provided by Dr Gang Pei (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) and Dr Justin McCarthy (University College Cork, Cork, Ireland). All vectors were confirmed by DNA sequencing. HEK-293T and A549 cells were transfected with siRNA oligonucleotides using Lipofectamine™ 2000. Two different oligonucleotides against USP4 were used: siRNA-1 (5′-TTAAACAGGTGGUGAGAAA-3′) and siRNA-2 (5′- CGAAGAATGGAGAGGAACA-3′).

Immunoprecipitation and immunoblotting

Immunoprecipitation was performed as described previously [18]. Briefly, transfected cells were lysed in lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% Nonidet P40 and a cocktail of protease inhibitors] and cleared by centrifugation (12000 g for 15 min at 4°C). Cleared cell lysates were incubated with 1 μg of anti-FLAG antibody (Sigma) and 16 μl of Protein A/G beads (Santa Cruz Biotechnology) for 3 h. To detect the interactions of endogenous proteins, HEK-293T cells were lysed in ice-cold lysis buffer. Cleared cell lysates were incubated with 3 μg of anti-USP4 (Bethyl Laboratories) and 16 μl of Protein A/G beads for 3 h at 4°C. After extensive washing, beads were boiled at 100°C for 5 min. Proteins were resolved by SDS/PAGE and transferred on to nitrocellulose membranes (Millipore) followed by immunoblotting using an anti-HA (Santa Cruz Biotechnology; 1:1000 dilution) or anti-FLAG (1:1000 dilution) antibody. Endogenous TRAF6, TRAF2 or USP4 was detected using anti-TRAF6 (Abcam; 1:1000 dilution), anti-TRAF2 (Santa Cruz Biotechnology; 1:1000 dilution) or anti-USP4 (1:1000 dilution) antibodies respectively. Immunoblots were analysed using the Odyssey system (LI-COR Biosciences).

Stable cell lines

The USP4 lentiviral vectors with GFP were co-transfected into HEK-293T cells with lentivirus packaging vectors using a calcium phosphate/DNA co-precipitation assay. Viral supernatants were collected after 48 h. Cells were incubated with lentiviral supernatant in the presence of 4 mg/ml polybrene. After incubation for 48 h, the cells were sorted for stable cell line analysis by flow cytometry.

GST pull-down assay

HEK-293T cells stably expressing USP4 were lysed and cleared by centrifugation (12000 g for 15 min at 4°C). Then, 10 μg of GST or GST–TRAF6 proteins purified from Escherichia coli were incubated with cell lysates for 1 h and the GST proteins were purified using glutathione–Sepharose 4B (Amersham Biosciences). The bound USP4 was detected by immunoblotting.

Luciferase assay

The indicated plasmids were transiently transfected into HEK-293T cells in the presence of NF-κB-dependent firefly luciferase and Renilla luciferase plasmids. The cells were harvested after 36 h and luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activity was calculated by dividing the firefly luciferase activity by the Renilla luciferase activity. Results represent three independent experiments performed in duplicate.

Deubiquitination assay

For in vivo deubiquitination, HEK-293T cells were transfected with His–ubiquitin in the presence of the plasmids as indicated. The transfected cells were lysed by denaturing buffer (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4 and 10 mM imidazole), followed by nickel bead purification. Ubiquitination was detected by immunoblot analysis. For the in vitro deubiquitination assay, FLAG–TRAF6 or FLAG–TRAF2 was co-transfected with His–ubiquitin into HEK-293T cells. At 24 h later, the transfected cells were stimulated with IL-1β or TNFα for 15 min. The cells were harvested using RIPA buffer [100 mM Tris/HCl (pH 7.4), 30 mM NaCl, 2.5% sodium deoxycholate, 2 mM EDTA and 2% Nonidet P40] containing protease inhibitor cocktail and NEM (N-ethylmaleimide). The cell lysates were incubated with FLAG M2 beads overnight with rotation at 4°C. After extensive washing with TBS [Tris-buffered saline (25 mM Tris/HCl, pH 7.4, 150 mM NaCl and 3 mM KCl)], FLAG–TRAF6 or FLAG–TRAF2 was eluted with elution buffer [1×PBS (pH 7.4)] containing 3×FLAG peptide. Then 15 μl of the eluted FLAG–TRAF6 or FLAG–TRAF2 was incubated with purified GST–USP4 in DUB assay buffer [50 mM Hepes/NaOH (pH 8.0), 10% glycerol and 3 mM DTT (dithiothreitol)] at 37°C for 4 h. Ubiquitination was analysed by Western blotting using an anti-His, anti-FLAG or anti-USP4 antibody.

RNA isolation and real-time RT–PCR

Total RNA was isolated from cells by using RNAiso Plus reagent (TaKaRa) as described in the manufacturer's protocol. For mRNA analysis, an aliquot containing 5 μg of total RNA was reverse-transcribed using the RevertAid First Stand cDNA synthesis kit (Fermentas). Specific primers were used to amplify cDNA. Real-time PCR was performed using the SYBR Green PCR master mix (Applied Biosystems). The primers for real-time RT–PCR were: GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (sense, 5′-GAGCTGAACGGGAAGCTCACTG-3′; and antisense, 5′-TGGTGCTCAGTGTAGCCCAGGA-3′); IL-6 (sense, 5′-CCTTCTCCACAAGCGCCTTC-3′; and antisense, 5′-GGCAAGTCTCCTCATTGAATC-3′); IL-8 (sense, 5′-TGATTGAGAGTGGACCACAC-3′; and antisense, 5′-AACTTCTCCACAACCCTCTG-3′); and COX2 (cyclo-oxygenase 2) (sense, 5′-GTGCTTAAACAGGAGCATCCT-3′; and antisense, 5′-GATAGCCACTCAAGTGTTGC-3′). The amplification protocol consisted of an initial 10 min denaturation step at 95°C, followed by 40 cycles of PCR at 95°C for 15 s and 60°C for 1 min, and this was followed by detection by the ABI Prism 7500 Sequence Detection System (Applied Biosystems). Relative expression levels were presented as the relative fold change and calculated using the formula: 2−ΔΔCt=2−(ΔCtSiUSP4−ΔCtcontrol) where each ΔCtCttarget−ΔCtGAPDH.

Migration assay

A cell migration assay was performed using Transwell® migration chambers (8 μm pore size; Millipore) according to the manufacturer's instructions. For each experiment, the number of cells in five random fields on the underside of the filter was counted, and three independent filters were analysed. For the wound healing assay, A549 cells were seeded and scratched with micropipette tips when the cells had grown to near confluency (~90%), and images were captured at 0 and 14 h after wounding. Cells were treated with mitomycin C (10 mg/ml; Sigma) to inhibit cell proliferation.

Live cell imaging

A549 cells were plated in 35 cm dishes and transfected as described above. At 48 h post-infection, images were captured every 5 min for 10 h using a Leica TCS SP5 confocal microscope and analysed using ImageJ (NIH) software.

RESULTS

USP4 associates with TRAF2 and TRAF6

To identify the novel regulator of TRAF6 E3 ubiquitin ligase, we used a co-immunoprecipitation assay to search for potential binding partners. Among the proteins (including USP8, USP25, USP28 and USP33) we examined, the DUB USP4 (also known as UnpEL/Unph) was found to specifically interact with TRAF6 in HEK-293T cells (Figure 1A). Moreover, TRAF2, another essential regulator of the NF-κB signalling pathway, could also bind to USP4. In contrast, USP4 failed to interact with TRAF3 under the same conditions, indicating that USP4 specifically binds to TRAF2 and TRAF6 (Figure 1A). The catalytically inactive mutant USP4 C311S [19] interacted with TRAF6 as well as wild-type USP4, indicating that the interaction between USP4 and TRAF6 is independent of the USP4 DUB activity (Figure 1B). To examine the interaction between USP4 and TRAF6 under more physiological conditions, endogenous USP4 was immunoprecipitated with an anti-USP4 antibody from HEK-293T cells and the associated TRAF6 was detected using an anti-TRAF6 antibody. As shown in Figure 1(C), endogenous TRAF6 was readily detected in USP4, but not in IgG, immunoprecipitates. In addition, endogenous USP4 was able to bind to TRAF6 in HEK-293T cells (Figure 1D). A GST pull-down assay showed that USP4 could bind to the purified GST–TRAF6 in vitro (Figure 1E). Taken together, these results indicate that USP4 is a novel binding partner of TRAF6.

USP4 associates with TRAF2 and TRAF6

Figure 1
USP4 associates with TRAF2 and TRAF6

(A) USP4 specifically interacted with TRAF2 and TRAF6. HA–USP4 (wild-type) was transfected into HEK-293T cells with empty vector or FLAG–TRAF2, FLAG–TRAF3 or FLAG–TRAF6. Transfected cells were harvested and immunoprecipitated with an anti-FLAG antibody, and the immunoprecipitates and the original whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. (B) USP4 associated with TRAF6-independent DUB activity. Wild-type HA–USP4 (WT) or C311S HA–USP4 (C/S) was transfected into HEK-293T cells with empty vector or FLAG–TRAF6. Binding was measured by immunoprecipitation assay. * indicates a non-specific band. (C) An endogenous interaction between USP4 and TRAF6. Endogenous USP4 was immunoprecipitated from HEK-293T cells using an anti-USP4 antibody, and the immunoprecipitates and original cell lysates were subjected to immunoblotting using anti-TRAF6 or anti-USP4 antibodies. (D) HEK-293T cells transfected with FLAG–TRAF6 were harvested and immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates and whole-cell extracts were immunoblotted with anti-USP4 or anti-FLAG antibodies. (E) GST or GST–TRAF6 proteins were purified from E. coli and incubated with the cell lysates stably expressing USP4. The products of GST pull-down were analysed by immunoblotting using anti-USP4 or anti-GST antibodies. The molecular mass in kDa is indicated on the right-hand side. IB, immunoblot; IP, immunoprecipitate; WCE, whole-cell extract; WT, wild-type.

Figure 1
USP4 associates with TRAF2 and TRAF6

(A) USP4 specifically interacted with TRAF2 and TRAF6. HA–USP4 (wild-type) was transfected into HEK-293T cells with empty vector or FLAG–TRAF2, FLAG–TRAF3 or FLAG–TRAF6. Transfected cells were harvested and immunoprecipitated with an anti-FLAG antibody, and the immunoprecipitates and the original whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. (B) USP4 associated with TRAF6-independent DUB activity. Wild-type HA–USP4 (WT) or C311S HA–USP4 (C/S) was transfected into HEK-293T cells with empty vector or FLAG–TRAF6. Binding was measured by immunoprecipitation assay. * indicates a non-specific band. (C) An endogenous interaction between USP4 and TRAF6. Endogenous USP4 was immunoprecipitated from HEK-293T cells using an anti-USP4 antibody, and the immunoprecipitates and original cell lysates were subjected to immunoblotting using anti-TRAF6 or anti-USP4 antibodies. (D) HEK-293T cells transfected with FLAG–TRAF6 were harvested and immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates and whole-cell extracts were immunoblotted with anti-USP4 or anti-FLAG antibodies. (E) GST or GST–TRAF6 proteins were purified from E. coli and incubated with the cell lysates stably expressing USP4. The products of GST pull-down were analysed by immunoblotting using anti-USP4 or anti-GST antibodies. The molecular mass in kDa is indicated on the right-hand side. IB, immunoblot; IP, immunoprecipitate; WCE, whole-cell extract; WT, wild-type.

USP4 contains a DUSP (domain present in USP) domain and an USP domain. The USP domain is conserved in most USP members and essential for its DUB activity [19]. To identify the domains of USP4 responsible for its interaction with TRAF6, we constructed serial deletion mutants of USP4 (Figure 2A). These deletion mutants were co-transfected with FLAG–TRAF6 into HEK-293T cells, and then the cell extracts were subjected to a co-immunoprecipitation assay. As shown in Figure 2(B), wild-type and the USP domain, but not the DUSP domain, of USP4 could bind to TRAF6 (Figure 2B), indicating that the C-terminal USP domain is responsible for the interaction of USP4 with TRAF6.

Domains responsible for the interaction between USP4 and TRAF6

Figure 2
Domains responsible for the interaction between USP4 and TRAF6

(A) The deletion mutants of USP4 used in the present study. (B) The USP domain of USP4 is responsible for its binding to TRAF6. Empty vector or FLAG–TRAF6 was co-transfected into HEK-293T cells with wild-type (WT) HA–USP4 or its deletion mutants respectively. Cell lysates were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates and whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. * indicates a non-specific band. (C) The deletion mutants of TRAF6 used in the present study. (D) The TRAF domain of TRAF6 is required for its binding to USP4. HA–USP4 was co-transfected into HEK-293T cells with control vector, FLAG-tagged full-length TRAF6, FLAG-tagged amino acids 1–347 of TRAF6, FLAG-tagged amino acids 120–347 of TRAF6 or FLAG-tagged amino acids 120–522 of TRAF6 respectively. Cell lysates were immunoprecipitated with an anti-FLAG antibody and the immunoprecipitates and whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. * indicates a non-specific band. The molecular mass in kDa is indicated on the right-hand side. IB, immunoblot; IP, immunoprecipitate; WCE, whole-cell extract; WT, wild-type.

Figure 2
Domains responsible for the interaction between USP4 and TRAF6

(A) The deletion mutants of USP4 used in the present study. (B) The USP domain of USP4 is responsible for its binding to TRAF6. Empty vector or FLAG–TRAF6 was co-transfected into HEK-293T cells with wild-type (WT) HA–USP4 or its deletion mutants respectively. Cell lysates were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitates and whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. * indicates a non-specific band. (C) The deletion mutants of TRAF6 used in the present study. (D) The TRAF domain of TRAF6 is required for its binding to USP4. HA–USP4 was co-transfected into HEK-293T cells with control vector, FLAG-tagged full-length TRAF6, FLAG-tagged amino acids 1–347 of TRAF6, FLAG-tagged amino acids 120–347 of TRAF6 or FLAG-tagged amino acids 120–522 of TRAF6 respectively. Cell lysates were immunoprecipitated with an anti-FLAG antibody and the immunoprecipitates and whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. * indicates a non-specific band. The molecular mass in kDa is indicated on the right-hand side. IB, immunoblot; IP, immunoprecipitate; WCE, whole-cell extract; WT, wild-type.

We also investigated the domains responsible for TRAF6 binding to USP4. TRAF6 contains three domains including an N-terminal RING-finger domain, a central zinc-finger domain and C-terminal TRAF domain [20] (Figure 2C). As shown in Figure 2(D), the mutant containing the zinc finger and TRAF domains retained the ability to bind to USP4, whereas the mutant containing amino acids 1–347 and 120–347 without the TRAF domain failed to bind to USP4. These results indicate that TRAF6 binds to USP4 through its TRAF domain.

USP4 inhibits NF-κB activation

Given the pivotal role for TRAF2 and TRAF6 in the NF-κB signalling pathway, we investigated whether USP4 is a negative regulator of TRAF2- and TRAF6-mediated NF-κB activation using an NF-κB-dependent luciferase reporter gene assay. As shown in Figures 3(A) and 3(B), co-expression of wild-type USP4 significantly suppressed TRAF6- and TRAF2-mediated NF-κB activation. In contrast, the USP4 C311S mutant without DUB activity reduced the inhibitory effect on NF-κB activation. These results suggest that DUB activity is responsible for the suppression of TRAF6- and TRAF2-mediated NF-κB activation by USP4. It has been reported previously that TRAF6 is involved in IL-1β-mediated NF-κB activation and that TRAF2 is involved in TNFα-mediated NF-κB activation [16]. Thus we examined whether USP4 can suppress TNFα- and IL-1β-induced NF-κB activation. As shown in Figure 3(C), ectopic expression of wild-type, but not the C311S mutant, USP4 suppressed both TNFα- and IL-1β-induced NF-κB activation. Previous studies indicate that TRAF2 is required for both TNFR1- and TNFR2-induced NF-κB activation [21]. Our results showed that USP4 could suppress both TNFR1- and TNFR2-mediated NF-κB activation (Figure 3D).

USP4 inhibits NF-κB activation

Figure 3
USP4 inhibits NF-κB activation

(A) USP4 suppressed TRAF6-mediated NF-κB activation in a USP4 DUB activity-dependent way. Empty vector or expression vectors encoding wild-type USP4 and the C311S USP4 mutant, along with the NF-κB-dependent firefly luciferase reporter and control Renilla luciferase reporter vectors, were co-transfected into HEK-293T cells. (B) USP4 suppressed TRAF2-mediated NF-κB activation. (C) USP4 suppressed the TNFα- and IL-1β-induced NF-κB activation in a DUB activity-dependent manner. Wild-type or C311S mutant USP4, along with NF-κB-dependent firefly luciferase reporter and control Renilla luciferase reporter vectors were transfected into HEK-293T cells. Transfected cells were stimulated with TNFα (10 ng/ml) or IL-1β (5 ng/ml). (D) USP4 suppressed TNFR1- and TNFR2-mediated NF-κB activation. (E) Knockdown efficiency was examined by immunoblotting using an anti-USP4 antibody. (F) Knockdown of endogenous USP4 promoted the NF-κB activation mediated by TRAF6. HEK-293T cells were transfected with control siRNA (siNC) or siRNA against USP4 (siUSP4). At 48 h later, cells were transfected with empty vector or expression vector encoding TRAF6. (G) Knockdown of endogenous USP4 promoted the NF-κB activation mediated by TRAF2. (H) Knockdown of endogenous USP4 promotes the NF-κB activation mediated by TNFR1 and TNFR2. (I) Knockdown of USP4 promotes the degradation of TNFα-induced IκBα. HEK-293T cells were transfected with control or USP4 siRNA and stimulated with TNFα (10 ng/ml) before harvest. The cell lysates were analysed by immunoblotting using anti-IκBα, anti-α-tubulin, anti-ERK (extracellular-signal-regulated kinase) or anti-USP4 antibodies respectively. (J) Knockdown of USP4 had little effect on TRAF2 and TRAF6 stability. HEK-293T cells were transfected with control or USP4 siRNA and stimulated with TNFα (10 ng/ml) for the time points indicated before harvesting. The cell lysates were analysed by immunoblotting using anti-IκBα, anti-TRAF2 anti-TRAF6, anti-actin or anti-USP4 antibodies respectively. WT, wild-type.

Figure 3
USP4 inhibits NF-κB activation

(A) USP4 suppressed TRAF6-mediated NF-κB activation in a USP4 DUB activity-dependent way. Empty vector or expression vectors encoding wild-type USP4 and the C311S USP4 mutant, along with the NF-κB-dependent firefly luciferase reporter and control Renilla luciferase reporter vectors, were co-transfected into HEK-293T cells. (B) USP4 suppressed TRAF2-mediated NF-κB activation. (C) USP4 suppressed the TNFα- and IL-1β-induced NF-κB activation in a DUB activity-dependent manner. Wild-type or C311S mutant USP4, along with NF-κB-dependent firefly luciferase reporter and control Renilla luciferase reporter vectors were transfected into HEK-293T cells. Transfected cells were stimulated with TNFα (10 ng/ml) or IL-1β (5 ng/ml). (D) USP4 suppressed TNFR1- and TNFR2-mediated NF-κB activation. (E) Knockdown efficiency was examined by immunoblotting using an anti-USP4 antibody. (F) Knockdown of endogenous USP4 promoted the NF-κB activation mediated by TRAF6. HEK-293T cells were transfected with control siRNA (siNC) or siRNA against USP4 (siUSP4). At 48 h later, cells were transfected with empty vector or expression vector encoding TRAF6. (G) Knockdown of endogenous USP4 promoted the NF-κB activation mediated by TRAF2. (H) Knockdown of endogenous USP4 promotes the NF-κB activation mediated by TNFR1 and TNFR2. (I) Knockdown of USP4 promotes the degradation of TNFα-induced IκBα. HEK-293T cells were transfected with control or USP4 siRNA and stimulated with TNFα (10 ng/ml) before harvest. The cell lysates were analysed by immunoblotting using anti-IκBα, anti-α-tubulin, anti-ERK (extracellular-signal-regulated kinase) or anti-USP4 antibodies respectively. (J) Knockdown of USP4 had little effect on TRAF2 and TRAF6 stability. HEK-293T cells were transfected with control or USP4 siRNA and stimulated with TNFα (10 ng/ml) for the time points indicated before harvesting. The cell lysates were analysed by immunoblotting using anti-IκBα, anti-TRAF2 anti-TRAF6, anti-actin or anti-USP4 antibodies respectively. WT, wild-type.

We also investigated the effect of endogenous USP4 on TRAF6-, TRAF2-, TNFR1- and TNFR2-mediated NF-κB activation. USP4 could be efficiently depleted by its specific siRNA (Figure 3E). As shown in Figure 3(F), knockdown of endogenous USP4 significantly increased TRAF2-, TRAF6-, TNFR1- and TNFR2-mediated NF-κB activation (Figures 3F–3H), suggesting that endogenous USP4 is also a negative regulator of TRAF6 and TRAF2 activity.

TNFα-stimulated NF-κB activation requires the ubiquitination-dependent degradation of IκBα [22]. Thus we tested whether USP4 regulates TNFα-induced IκBα degradation. As shown in Figures 3(I) and 3(J), knockdown of USP4 significantly promoted the degradation of IκBα induced by TNFα. In contrast, knockdown of USP4 had little effect on the stability of TRAF2 and TRAF6. Taken together, our results demonstrated that USP4 is a negative regulator of the NF-κB signalling pathway.

USP4 deubiquitinates TRAF2 and TRAF6

The enzymatic activity of TRAF6 requires its Lys63-linked polyubiquitination [11,16]. Therefore we investigated whether USP4 acts as a DUB targeting TRAF6 to inhibit NF-κB activation. FLAG–TRAF6 was transfected into HEK-293T cells with His–ubiquitin in the presence or absence of wild-type or C311S HA–USP4. The ubiquitinated TRAF6 was detected using an in vivo ubiquitination assay. As shown in Figure 4(A), wild-type USP4 markedly reduced the ubiquitination level of TRAF6, whereas the USP4 C311S mutant failed to do so. A similar inhibitory effect was also observed on TRAF2 (Figure 4A). In addition, we used an in vitro deubiquitination assay to confirm these results. In this assay, the FLAG–TRAF6 or FLAG–TRAF2 purified from HEK-239T cells were incubated with purified GST–USP4 and the level of ubiquitinated TRAF6 or TRAF2 was detected by Western blotting. We found that the presence of USP4 significantly reduced the ubiquitination of TRAF6 and TRAF2 (Figure 4B). The effect of endogenous USP4 on the ubiquitination of TRAF6 was also tested. In this assay, we found that knockdown of endogenous USP4 significantly increased the ubiquitination level of FLAG–TRAF6 (Figure 4C). Taken together, our results demonstrate that USP4 is a novel DUB that targets TRAF6 and TRAF2 for deubiquitination.

USP4 deubiquitinates TRAF2/TRAF6

Figure 4
USP4 deubiquitinates TRAF2/TRAF6

(A) USP4 deubiquitinated TRAF2/TRAF6. FLAG–TRAF2/TRAF6 was co-transfected with or without His–ubiquitin and HA–USP4 [wild-type (WT) or C311S mutant (CS)]. The ubiquitination of TRAF6 was measured using an in vivo ubiquitination assay. The precipitates and whole- cell extracts were analysed by immunoblotting using anti-FLAG or anti-HA antibodies. Ni-NTA indicates nickel bead precipitation. (B) USP4 deubiquitinated TRAF2/TRAF6 in vitro. HEK-293T cells were transfected with FLAG–TRAF2/TRAF6 and His–ubiquitin. Cell lysates were immunoprecipitated with an anti-FLAG antibody and then incubated with purified GST–USP4 proteins at 37°C for 4 h. Ubiquitination of TRAF2/TRAF6 was analysed by immunoblotting with an anti-His, anti-FLAG or anti-USP4 antibody. * indicates a non-specific band. (C) USP4 abrogated the endogenous ubiquitination of TRAF6. Control siRNA (siNC) or siRNA against USP4 (siUSP4) was transfected into HEK-293T cells. At 48 h later, cells were transfected with HA–ubiquitin and FLAG–TRAF6 and harvested after 24 h. Samples were immunoprecipitated with the anti-FLAG antibody, and the immunoprecipitates (IP) and the original whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. Cs, C311S mutant; IB, immunoblot; NSB, non-specific band; Ub, ubiquitin; WCE, whole-cell extract; WT, wild-type.

Figure 4
USP4 deubiquitinates TRAF2/TRAF6

(A) USP4 deubiquitinated TRAF2/TRAF6. FLAG–TRAF2/TRAF6 was co-transfected with or without His–ubiquitin and HA–USP4 [wild-type (WT) or C311S mutant (CS)]. The ubiquitination of TRAF6 was measured using an in vivo ubiquitination assay. The precipitates and whole- cell extracts were analysed by immunoblotting using anti-FLAG or anti-HA antibodies. Ni-NTA indicates nickel bead precipitation. (B) USP4 deubiquitinated TRAF2/TRAF6 in vitro. HEK-293T cells were transfected with FLAG–TRAF2/TRAF6 and His–ubiquitin. Cell lysates were immunoprecipitated with an anti-FLAG antibody and then incubated with purified GST–USP4 proteins at 37°C for 4 h. Ubiquitination of TRAF2/TRAF6 was analysed by immunoblotting with an anti-His, anti-FLAG or anti-USP4 antibody. * indicates a non-specific band. (C) USP4 abrogated the endogenous ubiquitination of TRAF6. Control siRNA (siNC) or siRNA against USP4 (siUSP4) was transfected into HEK-293T cells. At 48 h later, cells were transfected with HA–ubiquitin and FLAG–TRAF6 and harvested after 24 h. Samples were immunoprecipitated with the anti-FLAG antibody, and the immunoprecipitates (IP) and the original whole-cell extracts were analysed by immunoblotting using anti-HA or anti-FLAG antibodies. Cs, C311S mutant; IB, immunoblot; NSB, non-specific band; Ub, ubiquitin; WCE, whole-cell extract; WT, wild-type.

Evidence has shown that both TRAF2 and TRAF6 can be degraded by the ubiquitin–proteasome pathway [23,24]. Given that USP4 can deubiquitinate TRAF2 and TRAF6, we examined whether USP4 has any effect on their protein stability. Our results showed that neither overexpression nor knockdown of USP4 affected TRAF2 and TRAF6 protein stability (Supplementary Figures S1A and S1B at http://www.BiochemJ.org/bj/441/bj4410979add.htm). A previous study showed that cIAP is the major E3 ubiquitin ligase to degrade TRAF2 in the TNFR2 signalling pathway [23]. Therefore we examined whether USP4 affects cIAP-mediated TRAF2 degradation. Our results showed that USP4 has little effect on cIAP-mediated TRAF2 degradation (Supplementary Figure S1C). Taken together, the results suggest that USP4 regulates TRAF2- and TRAF6-mediated NF-κB activation mainly through the regulation of their polyubiquitination.

USP4 negatively regulates TNFα-induced gene expression

Given that USP4 is a negative regulator of TNFα- and TRAF2/TRAF6-mediated NF-κB activation, we investigated whether USP4 can regulate the TNFα-mediated biological function. Activation of NF-κB by TNFα can induce the expression of various genes, such as IL6 and IL8. Thus we asked whether USP4 is involved in TNFα-induced gene expression. USP4 has been suggested to be an oncoprotein which is elevated in several cancers, including small cell lung carcinomas and adenocarcinomas [25,26]. Our results have also shown that USP4 could be readily detected in cells of two lung cancer cell lines A549 and H1299 (results not shown). Specific siRNAs of USP4 could efficiently inhibit the expression of endogenous USP4 in A549 cells (Figure 5A). Moreover, knockdown of USP4 enhanced the TNFα-induced IκBα degradation in A549 cells, indicating that USP4 also negatively regulates TNFα-induced NF-κB activation in A549 cells (Figure 5B). To determine further the role of USP4 on NF-κB target gene expression, we extracted total RNAs from the control and USP4-knockdown A549 cells treated with TNFα for the indicated time points and performed quantitative RT–PCR to examine the effect of knockdown of USP4 on TNFα-induced IL6, IL8 and COX2. As shown in Figures 5(C)–5(E), knockdown of USP4 significantly enhanced the TNFα-induced expression of IL6, IL8 and COX2. Taken together, these results suggest that USP4 negatively regulates NF-κB target gene expression in cancer cells.

USP4 negatively regulates TNFα-mediated gene expression

Figure 5
USP4 negatively regulates TNFα-mediated gene expression

(A) Knockdown efficiency in A549 cells was examined by quantitative RT–PCR. (B) Knockdown of USP4 promoted the degradation of TNFα-induced IκBα. A549 cells were transfected with control or USP4 siRNA and stimulated with TNFα (10 ng/ml) before harvesting. The cell lysates were analysed by immunoblotting using anti-IκBα and anti-actin antibodies respectively. (C) A549 cells were transfected with control siRNA (siNC) or siRNA against USP4 (siUSP4). At 48 h later, the A549 cells were either untreated or treated with TNFα (10 ng/ml) for the time points indicated. Total RNAs from these cells were harvested. IL6 (C), IL8 (D) and COX2 (E) transcript levels in the siNC and two siUSP4 A549 cells were measured using quantitative RT–PCR and normalized to GAPDH. The data are presented as the mean±S.D. of three separate experiments.

Figure 5
USP4 negatively regulates TNFα-mediated gene expression

(A) Knockdown efficiency in A549 cells was examined by quantitative RT–PCR. (B) Knockdown of USP4 promoted the degradation of TNFα-induced IκBα. A549 cells were transfected with control or USP4 siRNA and stimulated with TNFα (10 ng/ml) before harvesting. The cell lysates were analysed by immunoblotting using anti-IκBα and anti-actin antibodies respectively. (C) A549 cells were transfected with control siRNA (siNC) or siRNA against USP4 (siUSP4). At 48 h later, the A549 cells were either untreated or treated with TNFα (10 ng/ml) for the time points indicated. Total RNAs from these cells were harvested. IL6 (C), IL8 (D) and COX2 (E) transcript levels in the siNC and two siUSP4 A549 cells were measured using quantitative RT–PCR and normalized to GAPDH. The data are presented as the mean±S.D. of three separate experiments.

USP4 inhibits TNFα-induced cancer cell migration

Previous results have shown that TNFα can promote the migration of breast cancer cells [27]. Interestingly, we found that TNFα could also significantly increase the migration of A549 cells, measured using a Transwell® migration assay (Figure 6A). Thus we asked whether USP4 affects TNFα-induced A549 cell migration. To answer this question, endogenous USP4 was knocked down by two siRNAs of USP4 and A549 cell migration was examined using a Transwell® migration assay. In this assay, we found that knockdown of USP4 markedly promoted TNFα-induced cell migration (Figure 6A). As expected, knockdown of USP4 also significantly increased IL-1β-stimulated cell migration (Figure 6B). This result was confirmed further using a wound healing assay. As shown in Figure 6(C), knockdown of USP4 promoted TNFα-induced wound healing in A549 cells. The effect of USP4 knockdown on A549 cell motility was also examined by time-lapse microscopy in the absence or presence of TNFα. We observed that knockdown of USP4 significantly enhanced both basal and TNFα-induced cell motility of A549 cells (Figure 6D). Taken together, our results indicate that USP4 is a negative regulator of TNFα-induced cell migration.

USP4 inhibits TNFα-induced cell migration

Figure 6
USP4 inhibits TNFα-induced cell migration

(A) Knockdown of USP4 promoted the TNFα-induced cancer cell migration. A549 cells transfected with control or USP4 siRNA were induced by TNFα (10 ng/ml) and the cell migration was examined using a Transwell® assay. Quantification is shown in the bottom panel. (B) Knockdown of USP4 promoted the IL-1β-induced cell migration. A549 cells transfected with control or USP4 siRNA were induced by IL-1β (10 ng/ml) and the cell migration was examined using a Transwell® assay. Quantification is shown in the bottom panel. (C) Knockdown of USP4 promoted the cell migration induced by TNFα measured using a wound healing assay. A549 cells transfected with control or USP4 siRNA were induced by TNFα and the cell migration ability was examined by wound healing assay (left-hand panel) with the quantification shown in the right-hand panel. (D) Depletion of USP4 enhanced A549 cell motility. A549 cells were transfected with control or USP4 siRNA and cell migration was recorded by time-lapse microscopy in the presence or absence of TNFα (10 ng/ml). The motility was calculated by ImageJ (NIH). Results are presented as means±S.D. *P<0.05; **P<0.01 (Student's t test).

Figure 6
USP4 inhibits TNFα-induced cell migration

(A) Knockdown of USP4 promoted the TNFα-induced cancer cell migration. A549 cells transfected with control or USP4 siRNA were induced by TNFα (10 ng/ml) and the cell migration was examined using a Transwell® assay. Quantification is shown in the bottom panel. (B) Knockdown of USP4 promoted the IL-1β-induced cell migration. A549 cells transfected with control or USP4 siRNA were induced by IL-1β (10 ng/ml) and the cell migration was examined using a Transwell® assay. Quantification is shown in the bottom panel. (C) Knockdown of USP4 promoted the cell migration induced by TNFα measured using a wound healing assay. A549 cells transfected with control or USP4 siRNA were induced by TNFα and the cell migration ability was examined by wound healing assay (left-hand panel) with the quantification shown in the right-hand panel. (D) Depletion of USP4 enhanced A549 cell motility. A549 cells were transfected with control or USP4 siRNA and cell migration was recorded by time-lapse microscopy in the presence or absence of TNFα (10 ng/ml). The motility was calculated by ImageJ (NIH). Results are presented as means±S.D. *P<0.05; **P<0.01 (Student's t test).

DISCUSSION

In the present study, we identified USP4 as a novel DUB targeting TRAF2/TRAF6. We provide the first evidence to show that USP4 directly binds to the TRAF domain to inhibit the TRAF2/TRAF6 activity by deubiquitinating in a DUB activity-dependent manner. In agreement with a previous study showing that USP4 down-regulates the NF-κB signalling pathway by targeting TAK1 [TGF (transforming growth factor)-β-activated kinase 1] [19], the present study also demonstrates that USP4 negatively regulates IL-1β- and TNFα-induced NF-κB activation. Thus, together with the previous study [19], we conclude that USP4 may negatively regulate the NF-κB signalling pathway by targeting multiple signalling molecules, including TRAF2, TRAF6 and TAK1. Although the precise mechanism underlying the regulation of NF-κB by USP4 under diverse physiological and pathological conditions is still unclear, targeting multiple signalling molecules suggests that USP4 is an important regulator of the NF-κB signalling pathway.

The present study demonstrates that USP4 is a negative regulator of TNFα-induced cell migration. TNFα is a key cytokine involved in inflammation, immunity and cellular homoeostasis. Increasing evidence also links the TNFα signalling pathway to tumorigenesis, including tumour transformation, cell proliferation, angiogenesis, invasion and metastasis in many cancers [28,29]. In the present study, we found TNFα can increase the migration of A549 cells, a human lung adenocarcinoma cell line. Importantly, our results showed that knockdown of endogenous USP4 promotes TNFα-induced cell migration (Figure 6A), indicating that USP4 is a negative regulator of cell migration. To our knowledge, this is the first report that a member of the USP DUB family is involved in TNFα-induced cancer cell migration.

The mechanism by which USP4 negatively regulates cell migration needs to be investigated further. A previous study has shown that TNFα may promote breast cancer cell migration by regulating the stability of Snail, a key regulator of EMT (epithelial–mesenchymal transition), via the NF-κB signalling pathway [27]. Whether the same mechanism is employed in A549 cells remains unknown. However, our unpublished results have shown that USP4 can promote the degradation of Snail (H. Li, N. Xiao and P. Wang, unpublished work). Thus whether USP4 regulates cell migration via promoting EMT is currently being investigated.

Growing evidence has shown that USP4 may be a potential oncoprotein [26]. For example, USP4 mRNA is elevated in various types of cancers, such as colon, thyroid and urinary cancer [26]. USP4 can promote tumorigenesis when overexpressed in mice [30]. It may inhibit the tumour suppressor p53 by stabilizing the E3 ubiquitin ligase ARF-BP1 [26]. However, reports have also shown the USP4 protein level to be decreased in lung cancer cell lines, and it is not elevated in breast and pancreatic cancer [26,31]. Moreover, USP4 is demonstrated as a negative regulator of the Wnt signalling pathway [32], which has tumorigenic activity [33]. The results of the present study suggest that USP4 is a negative regulator of cell migration of cancer cells. Cell migration is critical to cancer metastasis [34]. Thus further investigation is needed to examine the role of USP4 in tumorigenesis and metastasis.

Although TRAF6 is a critical E3 ubiquitin ligase to regulate the NF-κB signalling pathway, a variety of NF-κB-independent functions are also being uncovered. For example, TRAF6 can regulate TLR4 (Toll-like receptor 4)-induced autophagy by promoting the Lys63-linked ubiquitination of Beclin-1 [35]. It is also involved in TGFβ-induced activation of JNK (c-Jun N-terminal kinase) and p38 [36]. A recent study links TRAF6 to Huntington's disease by promoting the atypical ubiquitination of huntingtin protein [37]. Ubiquitination by TRAF6 is critical to membrane targeting and activation of Akt [38]. The present study shows that USP4 is a potential negative regulator of TRAF6 activity. Thus it will be of great interest to test whether USP4 is also involved in the NF-κB-independent function of TRAF6 by regulating the ubiquitination of other TRAF6 substrate targets.

It is still unclear whether USP4 activity is under the regulation of an extracellular signal to target TRAF2/TRAF6 and/or TAK1. Our results suggest that USP4 may constitutively associate with TRAF6 in vivo. Moreover, our unpublished results indicated that TRAF6 can promote the polyubiquitination of USP4 (H. Li, N. Xiao and P. Wang, unpublished work). Accumulating evidence has shown that Lys63-linked polyubiquitination can modulate the biological function of its target substrates, such as their activity and subcellular localization etc. [5,39]. Thus there is a possible model that USP4 may be activated by TRAF6-mediated ubiquitination and provides a negative-feedback loop to regulate TRAF2/TRAF6 and/or TAK1 ubiquitination.

In summary, in the present study we provide the first evidence that USP4 is a novel binding partner of TRAF2 and TRAF6, and acts as an essential DUB to inhibit NF-κB activation. Considering the results of the present study, and results of previous studies [10,11,13,16], we propose a working model (Figure 7), in which cytokines such as TNFα or IL-1β induce NF-κB activation, and polyubiquitination of TRAF6 or TRAF2 is critical to NF-κB activation. USP4 may rapidly deubiquitinate Lys63-linked ubiquitinated TRAF2/TRAF6 and prevents NF-κB activation, which then leads to the occurrence of regulated biological functions, such as cell migration.

Working model for the negative regulation of TRAF2/TRAF6 and NF-κB activation by USP4

Figure 7
Working model for the negative regulation of TRAF2/TRAF6 and NF-κB activation by USP4

Cytokines such as TNFα or IL-1β induce NF-κB activation, and the polyubiquitination of TRAF2/TRAF6 is critical to NF-κB activation. USP4 associates with TRAF2/TRAF6 and acts as a DUB to inhibit the ubiquitination of TRAF2/TRAF6 and consequently inhibits NF-κB activation. IL-1R, IL-1 receptor. K48, Lys48; K63, Lys63.

Figure 7
Working model for the negative regulation of TRAF2/TRAF6 and NF-κB activation by USP4

Cytokines such as TNFα or IL-1β induce NF-κB activation, and the polyubiquitination of TRAF2/TRAF6 is critical to NF-κB activation. USP4 associates with TRAF2/TRAF6 and acts as a DUB to inhibit the ubiquitination of TRAF2/TRAF6 and consequently inhibits NF-κB activation. IL-1R, IL-1 receptor. K48, Lys48; K63, Lys63.

Abbreviations

     
  • cIAP

    cellular inhibitor of apoptosis

  •  
  • COX2

    cyclo-oxygenase 2

  •  
  • DUB

    deubiquitinase

  •  
  • DUSP

    domain present in ubiquitin-specific protease

  •  
  • EMT

    epithelial–mesenchymal transition

  •  
  • FBS

    fetal bovine serum

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • GFP

    green fluorescent protein

  •  
  • GST

    glutathione transferase

  •  
  • HA

    haemagglutinin

  •  
  • HEK

    human embryonic kidney

  •  
  • IκB

    inhibitor of nuclear factor κB

  •  
  • IL

    interleukin

  •  
  • NF-κB

    nuclear factor κB

  •  
  • RT

    reverse transcription

  •  
  • siRNA

    small interfering RNA

  •  
  • TAK1

    TGF (transforming growth factor)-β-activated kinase 1

  •  
  • TNF

    tumour necrosis factor

  •  
  • TNFR

    TNF receptor

  •  
  • TRAF

    TNFR-associated factor

  •  
  • USP

    ubiquitin-specific protease

AUTHOR CONTRIBUTION

Ning Xiao and Ping Wang designed the research; Ning Xiao, Hui Li and Rui Wang performed the research; Ning Xiao, Hui Li, Haiquan Chen, Jiquan Chen and Ping Wang analysed the data; and Ning Xiao, Hui Li, Jiquan Chen and Ping Wang wrote the paper.

We thank Dr Gang Pei and Dr Justin McCarthy for providing reagents. We thank Dongmei Liu, Yunfei Chen, Taiqi Chen, Su Yu, Yingcong Wang and other members of the Wang laboratory for their assistance.

FUNDING

This work was supported, in part, by the National Basic Research Program of China [973 programme; grant numbers 2010CB529704 and 2012CB910400]; and the National Natural Science Foundation of China [grant numbers 30800587, 30971521 and 31171338]. P.W. is a scholar of the Shanghai Rising-Star Program from the Science and Technology Commission of Shanghai Municipality [grant number 09QA1401900].

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Author notes

1

These authors contributed equally to this work.

Supplementary data