UBL4A Augments Innate Immunity by Promoting the K63-Linked Ubiquitination of TRAF6 This information is current as Shu-Jie Peng, Ran-Ran Yao, Shuang-Shuang Yu, Hong-Yan of September 28, 2021. Chen, Xuewen Pang, Yu Zhang and Jun Zhang J Immunol 2019; 203:1943-1951; Prepublished online 26 August 2019; doi: 10.4049/jimmunol.1800750 http://www.jimmunol.org/content/203/7/1943 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/08/25/jimmunol.180075 Material 0.DCSupplemental http://www.jimmunol.org/ References This article cites 57 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/203/7/1943.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology UBL4A Augments Innate Immunity by Promoting the K63-Linked Ubiquitination of TRAF6 Shu-Jie Peng,1 Ran-Ran Yao,1 Shuang-Shuang Yu, Hong-Yan Chen, Xuewen Pang, Yu Zhang, and Jun Zhang Human UBL4A/GdX, encoding an ubiquitin-like protein, was shown in this study to be upregulated by viral infection and IFN stimulation. Then the functions of UBL4A in antiviral immune response were characterized. Overexpression of UBL4A promoted RNA virus–induced ISRE or IFN-b or NF-kB activation, leading to enhanced type I IFN transcription and reduced virus replication. Consistently, knockdown of UBL4A resulted in reduced type I IFN transcription and enhanced virus replication. Additionally, overexpression of UBL4A promoted virus-induced phosphorylation of TBK1, IRF3, and IKKa/b. Knockdown of UBL4A inhibited virus-induced phosphorylation of TBK1, IRF3, and IKKa/b. Coimmunoprecipitation showed that UBL4A interacted with TRAF6, and this interaction was enhanced upon viral infection. Ubiquitination assays showed that UBL4A Downloaded from promoted the K63-linked ubiquitination of TRAF6. Therefore, we reveal a novel positive feedback regulation of UBL4A in innate immune response combating virus invasion by enhancing the K63-linked ubiquitination of TRAF6. The Journal of Immunology, 2019, 203: 1943–1951. iral nucleic acids are sensed by pattern recognition re- promote the production of type I IFNs and proinflammatory ceptors (PRRs) and then induce the production of type I cytokines (24, 25). http://www.jimmunol.org/ V IFNs and proinflammatory cytokines (1, 2). For RNA TNF receptor–associated factor (TRAF) family members have viruses, TLR family member TLR3 senses dsRNA (3, 4), whereas been reported to be essential for the recruitment of the down- TLR7/8 senses ssRNA in the endosomes (5, 6). RIG-I–like re- stream kinases to the adaptor proteins. Among them, TRAF6 is a ceptors (RLRs) recognize viral dsRNA in the cytosol (7–9). For ubiquitin E3 ligase that attaches the K63-linked polyubiquitin DNA viruses, TLR9 senses unmethylated DNA in the endosomes chains on substrate proteins or itself (26). The autoubiquiti- (10). cGMP-AMP (cGAMP) synthase (cGAS) is the main iden- nation at Lys124 of TRAF6 is a key activation step for TRAF6 tified cytosolic viral DNA sensor (CDS) (2, 11). Besides, several (27). It is critical for the recruitment of IKKa/b/g and/or TBK other DNA sensors are reported, including AIM2, DAI, DDX41, complex to adaptor proteins (28, 29). By this, TRAF6 func- IFI16, RNA polymerase III, Lsm14A, etc. (12–17). Upon viral tions as an important signaling scaffold downstream of multiple by guest on September 28, 2021 invasion, viral nucleic acids are recognized by corresponding PRR such as TLRs, NLRs, RLRs, and CDSs, which eventually PRRs and then recruit the downstream adaptor proteins such as leads to the production of proinflammatory cytokines and type I MyD88, TRIF, MAVS, and STING (11, 18–20). Then, the acti- IFNs (19, 30–32). Therefore, the regulation at TRAF6 level vation signals transmitted by different sensors converge at may affect the activation of NF-kB signaling and multiple PRR the common kinase level IKKa/b/g and TBK1 (21–23). Then, signaling. phosphorylated by these kinases, the transcription factors NF-kB, Ubiquitin-like protein 4A (UBL4A)/X-linked gene in the IRF3, and IRF7 are activated and translocate into the nucleus and G6PD cluster at Xq28 (Gdx) was originally identified as a housekeeping gene that shares 43% identity to ubiquitin in the N terminus (33). However, the ubiquitin-related activity of UBL4A is not reported. UBL4A can form a complex with Bag6 and Department of Immunology, School of Basic Medical Sciences, NHC Key Laboratory Trc35 and localize to the endoplasmic reticulum (ER) to reg- of Medical Immunology, Ministry of Health (Peking University), Peking University Health Science Center, Beijing 100191, China ulate ER-associated degradation (34). UBL4A participates in the transport of tail-anchored proteins into the ER (35). UBL4A is 1These authors contributed equally to this work. also involved in DNA damage–induced cell death (36). UBL4A ORCIDs: 0000-0003-3074-3908 (S.-J.P.); 0000-0002-2733-0048 (R.-R.Y.). is a potential tumor suppressor by dephosphorylation of Stat3 Received for publication May 29, 2018. Accepted for publication July 24, 2019. via promoting the interaction between Tc45 and Stat3 (37). This work was supported by grants from the National Natural Science Foundation of For Akt activation, UBL4A mediates insulin-induced plasma- China (81873871 and 31470843) and the Nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT31039). membraned translocation of Akt. UBL4A is also an Arp2/3 Address correspondence and reprint requests to Dr. Jun Zhang, Peking University binding partner and mediates actin branching (38). Until now, Health Science Center, 38 Xue Yuan Road, Hai Dian District, Beijing 100191, China. the functions of UBL4A in antiviral immune response are not E-mail address: [email protected] characterized. The online version of this article contains supplemental material. In the current study, we report that UBL4A is a novel IFN- Abbreviations used in this article: CDS, cytosolic viral DNA sensor; ER, endoplas- stimulated gene (ISG). Overexpression of UBL4A promoted mic reticulum; iBMDM, immortalized bone marrow–derived macrophage; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; NC, negative control; PRR, pattern virus-induced signaling, whereas knockdown of UBL4A had the recognition receptor; RLR, RIG-I–like receptor; SeV, Sendai virus; siRNA, small opposite effects and promoted viral replication. Further studies interfering RNA; TRAF, TNF receptor–associated factor; UBL4A, ubiquitin-like showed that UBL4A physically associated with TRAF6 and protein 4A; VSV, vesicular stomatitis virus. then augment the antiviral immune response by promoting the Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 K63-linked ubiquitination of TRAF6. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800750 1944 POSITIVE REGULATION OF VIRUS-INDUCED SIGNALING BY UBL4A Materials and Methods Ab was added. Finally, ECL assay (ImageQuant LAS 500) was used to Cells, reagents, and plasmids detect the signal. HEK293, HEK293T, HeLa cells, and immortalized bone marrow–derived Coimmunoprecipitation macrophages (iBMDMs) were maintained in DMEM (Thermo Fisher HEK293T cells were seeded at a density of 2.0 3 105 cells/ml in a 10 cm Scientific, Waltham, MA) supplemented with 10% heat-inactivated FBS cell culture dish. After 24 h, the plasmids were transfected into cells. (CellMax) and penicillin (100 U/ml)/streptomycin (100 mg/ml). Lipofectamine The cells were harvested after another 24 h and then lysed for 1 h, and 3000 and Opti-MEM were purchased from Thermo Fisher Scientific. the supernatant was collected. Each sample was coincubated together Poly(I:C) and poly(dA:dT) were purchased from InvivoGen (San Diego, with the indicated Abs or mouse IgG for control overnight. The superna- CA). pENTER-Flag-His-UBL4A was purchased from Vigene Biosciences tant was coincubated together with protein A agarose beads for another (Rockville, MD). HA-tagged UBL4A was constructed by standard 2 h. Centrifugal pellets were washed with washing buffer. The results molecular biology techniques. Human IFN-a was from BioLegend, and were analyzed according to immunoblot analysis. For endogenous im- IFN-b was from PeproTech (Rocky Hill, NJ). munoprecipitation, HEK293 cells were treated with or without SeV infection for 24 h. Then, the cells were harvested, lysed, and immuno- Abs precipitated by IgG control or anti-UBL4A Ab. The blot was monitored Abs specific for p-TBK1, TBK1, p-IRF3, IRF3, p-IKKa/b, TRAF6, by anti-TRAF6 Ab. p-IkBa, and K63-linkage–specific polyubiquitin were purchased from Cell Signaling Technology (Danvers, MA). The TRAF6 Ab used for immu- In vivo ubiquitination assay noprecipitation was from Santa Cruz Biotechnology (Dallas,