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The ER-Localised Hrd1 Ubiquitinates and Inactivates Usp15 to Promote TLR4-Induced Inflammation During Bacterial Infection Yao Lu

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The ER-Localised Hrd1 Ubiquitinates and Inactivates Usp15 to Promote TLR4-Induced Inflammation During Bacterial Infection Yao Lu 1 The ER-localised Hrd1 ubiquitinates and inactivates Usp15 to promote 2 TLR4-induced inflammation during bacterial infection 3 Yao Lu1*, Ying Qiu1*, Peng Chen2, Haishuang Chang3, Luqiang Guo3, Fang Zhang4, Li 4 Ma1, Chi Zhang1, Xin Zheng1, Jun Xiao1, Ruiyue Zhong1, Lei Han1, Xiaoyan Xu1,5, 5 Yanbo Zhang1,6, Dangsheng Li1, Guisheng Zhong7, Rosemary Boyton8, Ying Huang3, 6 Yongning He3, Ronggui Hu2#, Bin Wei4,9#, Hongyan Wang1,10# 7 1 State Key Laboratory of Cell Biology, Key Laboratory of Systems Biology, CAS 8 Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and 9 Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 10 Innovation Center for Cell Signaling Network, Shanghai, 200031, China; 2 State Key 11 Laboratory of Molecule Biology, Key Laboratory of Systems Biology, CAS Center for 12 Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell 13 Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 14 Shanghai, 200031, China; 3 State Key Laboratory of Molecular Biology, National Center 15 for Protein Science Shanghai, Shanghai Science Research Center, Shanghai Key 16 Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell 17 Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of 18 Sciences, University of Chinese Academy of Sciences, Shanghai, 201210; 4 Wuhan 19 Institute of Virology, Chinese Academy of Sciences, Wuhan, China; 5 Experimental 20 Immunology Branch, National Cancer Institute, US National Institutes of Health, 21 Bethesda, Maryland, USA; 6 Division of Immunology, Department of Microbiology and 22 Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, 23 USA; 7 iHuman Institute, School of Life Science and Technology, ShanghaiTech 24 University, Shanghai, China; 8 Lung Immunology Group, Department of Infectious 25 Diseases, Faculty of Medicine, Imperial College London, London W12 0NN, United 26 Kingdom; 9 School of Life Sciences, Shanghai University, Shanghai 200444; 10 Cancer 27 Center, Shanghai Tenth People's Hospital, Tongji University, School of Medicine, 28 Shanghai 200072, China 29 1 30 *Contributed equally; #Corresponding author: [email protected]; 31 [email protected]; [email protected] 32 Running title: Hrd1 enhances TLR4 pathway upon infection 33 Keywords: Hrd1, Ubiquitination, Usp15, TLR4 pathway, Inflammation and Infection 34 35 ABSTRACT 36 The special organelle-located MAVS, STING and TLR3 are important for clearing viral 37 infections. Although TLR4 triggers NF-κB activation to produce proinflammatory 38 cytokines for bacteria clearance, effectors with special organelle localisation have not 39 been identified. Here, we screened over 280 E3 ubiquitin ligases and discovered that the 40 endoplasmic reticulum-located Hrd1 regulated TLR4-induced inflammation during 41 bacterial infection. Hrd1 directly interacted with the deubiquitinating enzyme (DUB) 42 Usp15. Unlike the classical function of Hrd1 in ER-associated degradation, Usp15 was 43 not degraded but lost its DUB activity for IκBα deubiquitination, resulting in excessive 44 NF-κB activation. Importantly, Hrd1 deficiency in macrophages protected mice against 45 LPS-induced septic shock, and knock-down of Usp15 in Hrd1 KO macrophages restored 46 the reduced IL-6 production. This study has proposed the crosstalk between Hrd1 and 47 TLR4 linking the ER-plasma membrane function during bacterial infection. 48 2 49 Introduction 50 Macrophages express pattern recognition receptors (PRRs) such as Toll-like 51 receptors (TLRs) and RIG-like receptors (RLRs) to sense pathogen-associated molecular 52 patterns (PAMPs) and trigger the innate immune response, leading to inflammation and 53 microbe clearance.1 The mitochondria-located MAVS (Mitochondrial antiviral-signaling 54 protein, also named VISA (virus-induced signaling adapter), IPS-1 and Cardif)2, 3, 4, 5, the 55 endoplasmic reticulum (ER)-located STING (Stimulator of interferon genes)6, 7, 8 and the 56 endosome-located TLR3 (Toll-like receptor 3)9 are important for type I IFN production 57 and clearance of viral infections4. Although the surface receptor TLR4-triggered NF-κB 58 activation is well studied for proinflammatory cytokine production and bacteria clearance, 59 downstream effectors with special organelle localisation have not been identified in the 60 TLR4 pathway. In recent years, studies of membrane contact sites within cells and their 61 role have been rapidly advancing, in particular insights have recently demonstrated the 62 contact sites exist and function between the largest organelle endoplasmic reticulum (ER) 63 and other organelles for cell homeostasis or disease pathogenesis by sensing the intra- or 64 extracellular stimulation 2, 3, 6, 10, 11. 65 While a balanced inflammatory response is pivotal to protecting the host against 66 microbes and self-injury, excessive activation of the TLR or RLR signalling pathway 67 could lead to serious inflammatory diseases, including septic shock or autoimmune 68 diseases. Septic shock is the most common cause of death in hospitalized patients and the 69 proinflammatory cytokine IL-6 is crucial in the pathophysiology of severe sepsis, and 70 IL-6 levels most significantly correlate with mortality rates compared to other cytokines12. 71 Accumulating studies have shown that E3 ubiquitin ligases are involved in TLR 3 72 signalling. TRAF6 (TNF receptor associated factor 6), a typical RING-type E3, can be 73 autoubiquitinated at Lys124, which is then recognized by the TAB (TGF-β activated 74 kinase 1 binding protein) 2/3 complex to further activate TAK1 (TGF-β activated kinase 75 1) and NEMO (NF-κB essential modulator). Pellino-1 increases LPS-driven Lys63-linked 76 polyubiquitination of IRAK1, TBK1 (TANK binding kinase 1) and TAK1 in TLR 77 signalling. Since E3 ligases and their substrates can be targeted to attenuate excessive 78 inflammation and sepsis, we were interested in investigating whether any E3 ligases with 79 special organelle localisation could be identified in the TLR4 pathway. 80 We screened over 280 E3 ligases using Dharmacon RNAi Screening Libraries and 81 identified Hrd1 as a RING-type E3 ubiquitin ligase, which positively regulated IL-6 82 production in LPS-treated macrophages. Hrd1, a homologue of yeast Hrd1p/Der3p13, 83 contains a transmembrane domain and is specifically located in the endoplasmic 84 reticulum (ER). The best-defined function of Hrd1 is to ubiquitinate misfolded/unfolded 85 proteins with help from other proteins in the ER-associated degradation (ERAD) 86 complex14, 15, 16, 17, which protects cells from ER-stress-induced apoptosis18. In agreement 87 with this role, Hrd1 (also known as Synoviolin, Syvn1) expression is enhanced in 88 synovial fibroblasts from rheumatoid arthritis (RA) patients19, and Hrd1+/− mice are 89 resistant to collagen-induced arthritis due to increased synovial cell apoptosis20, 21. 90 Previous studies have demonstrated that TLR4 is highly expressed in RA synovial tissue 91 lining and sublining macrophages22, and excessive levels of TNF-α and IL-6 accelerate 92 RA development. However, no knowledge is available about how Hrd1 affects 93 TLR4-induced inflammation. More importantly, the ER has a broad localisation 94 throughout the cell and can form direct physically contacts with the cell membrane11, 23. 4 95 We found that macrophages enhance ER membranes upon bacterial infection. Therefore, 96 it was interesting to further elucidate how the ER-located Hrd1 participates in 97 TLR4-induced inflammation in macrophages during bacterial infection. 98 This study has identified an ERAD-independent function of Hrd1 to increase 99 TLR4-induced proinflammatory cytokine production. We fished out Usp15 100 (Ubiquitin-specific protease 15) as a binding partner for Hrd1. Usp15 is a member of the 101 largest subfamily of cysteine protease DUBs (deubiquitinating enzymes). Prior studies 102 have indicated that Usp15 promotes cell survival by stabilizing IκBα in TNF-α stimulated 103 HeLa cells24, and Usp15 promotes type I interferon responses and pathogenesis during 104 neuroinflammation25. Other studies have demonstrated Usp15 function in anti-tumor 105 response, including that Usp15 regulates p53 function to promote cancer-cell survival and 106 Usp15 inhibits T cell activation and immune surveillance26. However, it remains unclear 107 about how Usp15 affects TLR4-induced inflammation. This study has demonstrated that 108 Hrd1 promoted polyubiquitination of Lys21 in Usp15. Unlike other Hrd1 substrates, 109 ubiquitinated Usp15 was not degraded, but rather lost its DUB activity and failed to 110 deubiquitinate IκBα, which resulted in excessive TLR4-NF-κB activation. 111 Results 112 RNAi screening identifies the ER-localised Hrd1 to positively regulate inflammation 113 in LPS-stimulated macrophages 114 To identify E3 ligases involved in the regulation of TLR4-signalling, we transfected 115 over 280 siRNAs (Dharmacon RNAi Screening Libraries) into murine primary peritoneal 116 exudate macrophages (PEMs), followed by LPS stimulation for 6 hr. IL-6 concentrations 5 117 in supernatants were measured by ELISA (Figure S1a).We controlled macrophage 118 survival by checking the am-blue absorbance and excluded genes that significantly 119 induced macrophage death (indicated by blue colour in Figure 1a). Approximately 20 120 candidate genes were identified to affect IL-6 production, including Birc2 and Traf5 that 121
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