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ER-localized Hrd1 ubiquitinates and inactivates Usp15 to promote TLR4-induced inflammation during bacterial infection

Nature Microbiology volume 4, pages 2331–2346 (2019)Cite this article

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Abstract

The special organelle-located MAVS, STING and TLR3 are important for clearing viral infections. Although TLR4 triggers NF-κB activation to produce pro-inflammatory cytokines for bacterial clearance, effectors with special organelle localization have not been identified. Here, we screened more than 280 E3 ubiquitin ligases and discovered that the endoplasmic reticulum-located Hrd1 regulates TLR4-induced inflammation during bacterial infection. Hrd1 interacts directly with the deubiquitinating enzyme Usp15. Unlike the classical function of Hrd1 in endoplasmic reticulum-associated degradation, Usp15 is not degraded but loses its deubiquitinating activity for IκBα deubiquitination, resulting in excessive NF-κB activation. Importantly, Hrd1 deficiency in macrophages protects mice against lipopolysaccharide-induced septic shock, and knockdown of Usp15 in Hrd1-knockout macrophages restores the reduced IL-6 production. This study proposes that there is crosstalk between Hrd1 and TLR4, thereby linking the endoplasmic reticulum–plasma membrane function during bacterial infection.

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Fig. 1: ER-localized Hrd1 was identified by RNAi screening to positively regulate inflammation in LPS-stimulated macrophages.
Fig. 2: Hrd1-cKO macrophages specifically reduce TLR4-induced inflammation and NF-κB activation.
Fig. 3: Hrd1 E3-ligase activity is critical for TLR4-induced NF-κB activation independent of ERAD.
Fig. 4: ER-localized Hrd1 directly binds Usp15 and regulates TLR4-induced inflammation.
Fig. 5: Hrd1 promotes the polyubiquitination of Lys21 in Usp15 and inactivates Usp15.
Fig. 6: Hrd1 deficiency protects against LPS- and CLP-induced septic shock.

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Data availability

The original data that support the findings of this study are available from the corresponding author on request. The Supplementary figures are available in the Supplementary information section.

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Acknowledgements

We thank S. Sun, C. Wang and B. Sun for providing plasmids, A. Lin for the MEF cell lines, W. H. Fang for S. typhimurium (strain SL1344), Y. He for the Lysozyme M (LysM)–Cre+/+ mice and P. Wang for the DUb-7-amido-4-methylcoumarin reagents. We would like to thank the Core Facility of Chemical Biology and Core Facility of Molecular Biology for technical assistance, the National Center for Protein Science Shanghai for the mass spectrometry analysis and electron-microscopy data collection. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB19000000), the Ministry of Science and Technology of China (grant nos. 2016YFD0500207, 2018YFA0800702, 2016YFD0500407 and 2016YFC0905902), the National Natural Science Foundation of China (grant nos. 81825011, 81630043, 81571617, 81671572, 81571552, 81701569, 31700781 and 81801575) and the State Key Laboratory of Cell Biology, SIBCB, CAS (grant no. SKL CBKF2013003). We thank the Genome Tagging Project Center, Shanghai Institute of Biochemistry and Cell Biology, CAS for technical support. H.W. is supported by the Hundred Talents Program of the Chinese Academy of Sciences.

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  1. These author contributed equally: Yao Lu, Ying Qiu.

Authors and Affiliations

  1. State Key Laboratory of Cell Biology, Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Innovation Center for Cell Signaling Network, Shanghai, China

    Yao Lu, Ying Qiu, Li Ma, Chi Zhang, Xin Zheng, Jun Xiao, Ruiyue Zhong, Lei Han, Xiaoyan Xu, Yanbo Zhang, Dangsheng Li & Hongyan Wang

  2. State Key Laboratory of Molecule Biology, Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Peng Chen & Ronggui Hu

  3. State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Science Research Center, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Haishuang Chang, Luqiang Guo, Ying Huang & Yongning He

  4. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

    Fang Zhang & Bin Wei

  5. School of Life Sciences, Shanghai University, Shanghai, China

    Fang Zhang & Bin Wei

  6. Experimental Immunology Branch, National Cancer Institute, US National Institutes of Health, Bethesda, MA, USA

    Xiaoyan Xu

  7. Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA

    Yanbo Zhang

  8. iHuman Institute, School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Guisheng Zhong

  9. Lung Immunology Group, Adult Infectious Disease, Department of Infectious Diseases, Faculty of Medicine, Imperial College London, London, UK

    Rosemary Boyton

  10. Department of Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, UK

    Rosemary Boyton

  11. Cancer Center, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai, China

    Bin Wei & Hongyan Wang

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Contributions

Y.L. and Y.Q. performed the majority of experiments and statistical analyses with the help of H.C., L.G., F.Z., L.M., C.Z., X.Z., J.X., R.Z., L.H., X.X., Y.Z. and P.C. participated in part of the ubiquitination experiments. Y.Huang generated the interaction model of Hrd1 and Usp15. H.W., B.W., D.L., Y.L., Y.Q., G.Z., R.B., Y.He and R.H. designed the study. Y.L. and H.W. drafted the manuscript.

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Correspondence to Ronggui Hu, Bin Wei or Hongyan Wang.

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Lu, Y., Qiu, Y., Chen, P. et al. ER-localized Hrd1 ubiquitinates and inactivates Usp15 to promote TLR4-induced inflammation during bacterial infection. Nat Microbiol 4, 2331–2346 (2019). https://doi.org/10.1038/s41564-019-0542-2

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