HSCARG Negatively Regulates the Cellular Antiviral RIG-I Like Receptor Signaling Pathway by Inhibiting TRAF3 Ubiquitination <i>via</i> Recruiting OTUB1

<div><p>RIG-I like receptors (RLRs) recognize cytosolic viral RNA and initiate innate immunity; they increase the production of type I interferon (IFN) and the transcription of a series of antiviral genes to protect the host organism. Accurate regulation of the RLR pathway is important for avoiding tissue injury induced by excessive immune response. HSCARG is a newly reported negative regulator of NF-κB. Here we demonstrated that HSCARG participates in innate immunity. HSCARG inhibited the cellular antiviral response in an NF-κB independent manner, whereas deficiency of HSCARG had an opposite effect. After viral infection, HSCARG interacted with tumor necrosis receptor-associated factor 3 (TRAF3) and inhibited its ubiquitination by promoting the recruitment of OTUB1 to TRAF3. Knockout of HSCARG attenuated the de-ubiquitination of TRAF3 by OTUB1, and knockdown of OTUB1 abolished the effect of HSCARG. HSCARG also interacted with Ikappa-B kinase epsilon (IKKε) after viral infection and impaired the association between TRAF3 and IKKε, which further decreased the phosphorylation of IKKε and interferon response factor 3 (IRF3), thus suppressed the dimerization and nuclear translocation of IRF3. Moreover, knockdown of TRAF3 dampened the inhibitory effect of <i>IFN-β</i> transcription by HSCARG, suggesting that TRAF3 is necessary for HSCARG to down-regulate RLR pathway. This study demonstrated that HSCARG is a negative regulator that enables balanced antiviral innate immunity.</p></div

HSCARG and OTUB1 cooperatively inhibit TRAF3 ubiquitination.

<p>(A) HSCARG interacts with OTUB1. HEK293T cells transfected with indicated plasmids were infected with or without SeV (40 HAU/ml) for 6 h. Co-IP analysis was performed with anti-Flag or anti-Myc antibody followed by IB with anti-HA antibody. (B) HSCARG promotes TRAF3-OTUB1 interaction. <i>HSCARG</i>^−/− and wild-type HEK293T cells were transfected with Flag-TRAF3, HA-OTUB1, or together with Myc-HSCARG. Co-IP was then performed with anti-Flag and IB with anti-HA to examine the effect of HSCARG on the TRAF3-OTUB1 complex. Quantification of HA-OTUB1 was performed using Odyssey Infrared Imaging System and software Odyssey V3.0. The data was normalized to the enriched protein. (C) OTUB1 predominantly loses its de-ubiquitination ability in <i>HSCARG</i>^−/− cells. The wild-type and <i>HSCARG</i>^−/− HEK293T cells were transfected with HA-TRAF3, His-ubiquitin, together with or without Flag-OTUB1, and then subjected to His-ubiquitin pull-down analysis and IB with anti-HA to measure TRAF3 ubiquitination level. (D) HSCARG relies on OTUB1 to inhibit TRAF3 ubiquitination. HEK293T cells were transfected with OTUB1 siRNA (40 nM), negative control, and HA-TRAF3, His-ubiquitin with or without Myc-HSCARG as indicated. 72 h later, His-ubiquitin pull-down was performed followed by IB with anti-HA to detect TRAF3 ubiquitination level.</p

Working model of TRAF3-dependent regulation of RLR signaling pathway by HSCARG.

<p>Followed viral infection, HSCARG interacts with TRAF3, and cooperates with OTUB1 to remove Lys63-linked polyubiquitin chain from TRAF3. This further decreases the recruitment of IKKε and results in decreased phosphorylation of IKKε and IRF3, and finally reduces the production of IFN-β.</p

HSCARG interacts with IKKε and blocks the formation of TRAF3-IKKε complex.

<p>(A) Endogenous HSCARG interacts with IKKε. HEK293T cells transfected with Flag-IKKε were IP with anti-Flag followed by IB with anti-HSCARG or anti-Flag. (B, C) HSCARG blocks the interaction between IKKε and TRAF3. HEK293T cells transfected with Flag-IKKε, HA-TRAF3 with or without Myc-HSCARG (B), or HSCARG shRNA (C) were subjected to Co-IP analysis to examine the effect of HSCARG on the IKKε-TRAF3 interaction. (D) HSCARG decreases IKKε and IRF3 phosphorylation <i>in vitro</i>. Flag-IKKε was enriched from cell lysate with anti-Flag, GST, GST-IRF3 (131–426) and His-HSCARG were purified from <i>E.coli</i>, and <i>in vitro</i> phosphorylation was performed as described in the Methods. Anti-TRAF3 antibody was used to detect the existence of TRAF3 in the enriched Flag-IKKε. (E) HSCARG impairs IRF3 phosphorylation. HEK293T cells transfected with vector or Flag-RNF5 or Flag-HSCARG were infected with SeV for 6 h, and then IRF3 phosphorylation was analyzed with p-IRF3 antibody. (F) HSCARG suppresses IRF3 dimerization. HEK293T cells transfected with control vector or Flag-RNF5 or Flag-HSCARG were infected with SeV for 6 h, and then the effect of HSCARG on IRF3 dimerization was examined by Native PAGE. (G) HSCARG inhibits the nuclear translocation of TRAF3 triggered by viral infection. The wild-type and <i>HSCARG^−/</i>⁻ HeLa cells were infected by SeV (40 HAU/ml) for 0 and 6 h, and the subcellular location of endogenous IRF3 was detected with anti-IRF3 using Zeiss LSM 710&NLO. Scale bar, 10 µm.</p

HSCARG interacts with TRAF3 and inhibits TRAF3 K63-linked ubiquitination.

<p>(A) HSCARG interacts with endogenous TRAF3. HEK293T cells were transfected with Flag-HSCARG for 24 h, and then infected with SeV (40 HAU/ml) for 6 h. Cell lysates were IP with anti-Flag or control IgG followed by IB with antibodies against TRAF3 and Flag. (B) Mapping of the TRAF3 domains for HSCARG binding. HEK293T cells were transfected with HA-HSCARG and Flag-TRAF3 1–108, 109–347, 348–568 truncation constructs, Co-IP was performed with anti-Flag antibody followed by IB with anti-HA antibody. The protein expression level was shown in the bottom. A diagram for TRAF3 truncations was shown on top. (C) Viral infection promotes endogenous TRAF3 ubiquitination. HEK293T cells were transfected with His-ubiquitin and infected with SeV for 6 h, and then His-ubiquitin pull-down analysis was performed followed by IB with antibody against TRAF3 to examine the level of endogenous TRAF3 ubiquitination. (D, E) TRAF3 is mainly modified with K63-linked ubiquitination after viral infection. HEK293T cells were transfected with HA-TRAF3, His-ubiquitin, together with or without Flag-HSCARG (D), or with various ubiquitin mutants <i>K63R</i>, <i>K48R</i>, <i>K63</i>, and <i>K48</i> (E). 24 h later, cells were treated with MG132 (20 ng/ml) and SeV (40 HAU/ml) for 6 h (D), and then subjected to His-ubiquitin pull-down and IB with anti-HA to monitor the ubiquitination of TRAF3. Here, <i>K63R</i> or <i>K48R</i> means that only Lysine63 or Lysine48 residue was mutated to arginine, <i>K63</i> or <i>K48</i> means only Lysine63 or Lysine48 residue remains unchanged while the other lysines were all mutated to arginines. (F) HSCARG inhibits TRAF3 ubiquitination. Wild-type and <i>HSCARG</i>^−/− HEK293T cells transfected with Flag-TRAF3, His-ubiquitin, or in combination with HA-HSCARG were subjected to His-ubiquitin pull-down and IB with anti-Flag to examine the effect of HSCARG on TRAF3 ubiquitination.</p

HSCARG negatively regulates cellular antiviral response.

<p>(A) HSCARG down-regulates <i>IFN-β</i> reporter activity. HEK293T cells (1×10⁵) were transfected with 200 ng <i>IFN-β</i> reporter plasmid, 20 ng pRL-TK1 control plasmid, and increasing dose of HSCARG (200, 400 and 800 ng), the positive control PCBP2 (200, 400 and 800 ng), or HSCARG shRNA plasmid (300 and 600 ng), and infected with SeV (40 HAU/ml) for 18 h, and then luciferase reporter assay was performed. The induction folds are presented relative to the luciferase activity in the control cells transfected with the empty vector. (B) HSCARG suppresses the <i>IFN-β</i> activity mediated by the RLR adaptors. HEK293T cells transfected with plasmids encoding RIG-I (N terminal), MDA5 (N terminal), MAVS, TBK1, IKKε, IRF7, IRF3, with vector or increasing dose of HSCARG, were infected with SeV (40 HAU/ml) for 18 h, and then the <i>IFN-β</i> reporter activity was examined. (C) The induction of <i>IFN-β</i> is increased in <i>HSCARG</i>^−/− HEK293T. The wild-type and <i>HSCARG</i>^−/− cells were transfected with the same amount of plasmids encoding RIG-I N (N terminal), MDA5-N (N terminal), MAVS, TBK1, IKKε, IRF3, and the <i>IFN-β</i> reporter activity was assessed as described above. (D) SeV infection induces more mRNA of <i>IFN-β</i> in <i>HSCARG</i>^−/− HCT116 cells. Cells (1×10⁵) were harvested at 0, 24, and 48 h after SeV infection and RT-PCR was performed to monitor the <i>IFN-β</i> mRNA levels. The band of <i>HSCARG</i> mRNA in the <i>HSCARG</i>^−/− cells is larger and thus its mobility is slower in the gel compared to the native <i>HSCARG</i> mRNA. (E) HSCARG suppresses IFN-β secretion in media supernatant. HEK293T cells (1×10⁵) transfected with increasing dose of HSCARG (left panel), or indicated plasmids together with vector or HSCARG (right panel) were treated with SeV (40 HAU/ml) for 18 h, and then ELISA was performed to detect the secreted IFN-β level in supernatant. (F) HSCARG promotes VSV proliferation. HEK293T cells (1×10⁵) were transfected with indicated plasmids as shown in the horizontal axis with or without HSCARG. 24 h later, cells were infected with VSV at MOI = 1 for 1 h, and then plaque assay was performed to measure virus titer. Experiments were performed at least three times with similar results. The bar graphs show mean±S.D. of triplicates from one representative experiment. *<i>p</i><0.05, **<i>p</i><0.01.</p

HSCARG and OTUB1 cooperatively inhibit TRAF3 ubiquitination.

<p>(A) HSCARG interacts with OTUB1. HEK293T cells transfected with indicated plasmids were infected with or without SeV (40 HAU/ml) for 6 h. Co-IP analysis was performed with anti-Flag or anti-Myc antibody followed by IB with anti-HA antibody. (B) HSCARG promotes TRAF3-OTUB1 interaction. <i>HSCARG</i>^−/− and wild-type HEK293T cells were transfected with Flag-TRAF3, HA-OTUB1, or together with Myc-HSCARG. Co-IP was then performed with anti-Flag and IB with anti-HA to examine the effect of HSCARG on the TRAF3-OTUB1 complex. Quantification of HA-OTUB1 was performed using Odyssey Infrared Imaging System and software Odyssey V3.0. The data was normalized to the enriched protein. (C) OTUB1 predominantly loses its de-ubiquitination ability in <i>HSCARG</i>^−/− cells. The wild-type and <i>HSCARG</i>^−/− HEK293T cells were transfected with HA-TRAF3, His-ubiquitin, together with or without Flag-OTUB1, and then subjected to His-ubiquitin pull-down analysis and IB with anti-HA to measure TRAF3 ubiquitination level. (D) HSCARG relies on OTUB1 to inhibit TRAF3 ubiquitination. HEK293T cells were transfected with OTUB1 siRNA (40 nM), negative control, and HA-TRAF3, His-ubiquitin with or without Myc-HSCARG as indicated. 72 h later, His-ubiquitin pull-down was performed followed by IB with anti-HA to detect TRAF3 ubiquitination level.</p

TRAF3 is necessary for HSCARG to down-regulate <i>IFN-β</i>.

<p>HEK293T wild-type cells and TRAF3 depleted cells (two stable cell lines harboring TRAF3 shRNA were obtained, or cells treated with 40 nM TRAF3 siRNA) were transfected with 200 ng <i>IFN-β</i> reporter and 20 ng pRL-TK1 plasmids, together with 600 ng vector or Flag-HSCARG, and then infected with SeV for 12 h. Luciferase reporter assay was performed to examine the activation of <i>IFN-β</i> in cells with depleted TRAF3. The knockdown effect of TRAF3 was shown at the bottom. Experiments were performed in triplicates for at least three times. The bar graphs show mean±S.D. **<i>p</i><0.01.</p

Patient details.

<p>^a Fisher exact test. ASA, American Society of Anesthesiologists Class; pTNM, tumor, node and metastasis; BMI, body mass index</p><p>Patient details.</p

Long-Term Oncologic Outcomes of Laparoscopic versus Open Surgery for Middle and Lower Rectal Cancer - Fig 3

<p>A. Comparison of the 5-year overall recurrence of patients between laparoscopic and open groups (log-rank = 0.012; <i>P</i> = 0.913,). No. at risk: Laparoscopic group: 113.0\111.0\106.0\97.0\91.0\45.0;Open group: 123.0\119.0\110.0\104.0\100.0\49.5. B. Comparison of the 5-year overall survival in patients of different stages between laparoscopic and open group (P = 0.004).</p