Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors

  • An Erratum to this article was published on 01 September 2004


Activation of Toll-like receptors (TLRs) results in a proinflammatory response needed to combat infection. Thus, limiting TLR signaling is essential for preventing a protective response from causing injury to the host. Here we describe how a RING finger protein, Triad3A, acts as an E3 ubiquitin-protein ligase and enhances ubiquitination and proteolytic degradation of some TLRs. Triad3A overexpression promoted substantial degradation of TLR4 and TLR9 with a concomitant decrease in signaling, but did not affect TLR2 expression or signaling. Conversely, a reduction in endogenous Triad3A by small interfering RNA increased TLR expression and enhanced TLR activation. Thus, ubiquitination by Triad3A represents one pathway by which the intensity and duration of TLR signaling is controlled.

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Figure 1: Interaction between Triad3 and TLRs.
Figure 2: Structure, distribution, and protein sequence of Triad3A.
Figure 3: Triad3A is an E3 ubiquitin-protein ligase.
Figure 4: Triad3A regulates degradation of TLRs.
Figure 5: Triad3A negatively regulates TLRs activation in multiple cell types.
Figure 6: Depletion of Triad3A enhances TLR expression and cellular responses to TLR ligands.

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  1. 1

    Chuang, T.H. & Ulevitch, R.J. Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells. Biochim. Biophys. Acta 1518, 157–161 (2001).

    CAS  Article  Google Scholar 

  2. 2

    Aderem, A. & Ulevitch, R.J. Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787 (2000).

    CAS  Article  Google Scholar 

  3. 3

    Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Sabroe, I., Parker, L.C., Wilson, A.G., Whyte, M.K. & Dower, S.K. Toll-like receptors: their role in allergy and non-allergic inflammatory disease. Clin. Exp. Allergy 32, 984–989 (2002).

    CAS  Article  Google Scholar 

  5. 5

    Zuany-Amorim, C., Hastewell, J. & Walker, C. Toll-like receptors as potential therapeutic targets for multiple diseases. Nat. Rev. Drug Discov. 1, 797–807 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Bochud, P.Y. & Calandra, T. Pathogenesis of sepsis: new concepts and implications for future treatment. BMJ 326, 262–266 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Sweet, M.J. et al. A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression. J. Immunol. 166, 6633–6639 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Wald, D. et al. SIGIRR, a negative regulator of Toll-like receptor–interleukin 1 receptor signaling. Nat. Immunol. 4, 920–927 (2003).

    CAS  Article  Google Scholar 

  9. 9

    Kobayashi, K. et al. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 110, 191–202 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Nakagawa, R. et al. SOCS-1 participates in negative regulation of LPS responses. Immunity. 17, 677–687 (2002).

    CAS  Article  Google Scholar 

  11. 11

    Pickart, C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 82, 373–428 (2002).

    CAS  Article  Google Scholar 

  13. 13

    Strous, G.J. & Gent, J. Dimerization, ubiquitylation and endocytosis go together in growth hormone receptor function. FEBS Lett. 529, 102–109 (2002).

    CAS  Article  Google Scholar 

  14. 14

    Dikic, I. & Giordano, S. Negative receptor signalling. Curr. Opin. Cell Biol. 15, 128–135 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Daun, J.M. & Fenton, M.J. Interleukin-1/Toll receptor family members: receptor structure and signal transduction pathways. J. Interferon Cytokine Res. 20, 843–855 (2000).

    CAS  Article  Google Scholar 

  16. 16

    O'Neill, L.A., Fitzgerald, K.A. & Bowie, A.G. The Toll-IL-1 receptor adaptor family grows to five members. Trends Immunol. 24, 286–290 (2003).

    Article  Google Scholar 

  17. 17

    Janssens, S. & Beyaert, R. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol. Cell 11, 293–302 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Barton, G.M. & Medzhitov, R. Linking Toll-like receptors to IFN-α/β expression. Nat. Immunol. 4, 432–433 (2003).

    CAS  Article  Google Scholar 

  19. 19

    van der Reijden, B.A., Erpelinck-Verschueren, C.A., Lowenberg, B. & Jansen, J.H. TRIADs: a new class of proteins with a novel cysteine-rich signature. Protein Sci. 8, 1557–1561 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Chen, D., Li, X., Zhai, Z. & Shu, H.B. A novel zinc finger protein interacts with receptor-interacting protein (RIP) and inhibits tumor necrosis factor (TNF)- and IL1-induced NF-κB activation. J. Biol. Chem. 277, 15985–15991 (2002).

    CAS  Article  Google Scholar 

  21. 21

    Shimura, H. et al. Ubiquitination of a new form of α-synuclein by parkin from human brain: implications for Parkinson's disease. Science 293, 263–269 (2001).

    CAS  Article  Google Scholar 

  22. 22

    Chung, K.K. et al. Parkin ubiquitinates the α-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease. Nat. Med. 7, 1144–1150 (2001).

    CAS  Article  Google Scholar 

  23. 23

    Zhang, Y. et al. Parkin functions as an E2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1. Proc. Natl. Acad. Sci. USA 97, 13354–13359 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Niwa, J. et al. Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity. J. Biol. Chem. 277, 36793–36798 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Moynihan, T.P. et al. The ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1. J. Biol. Chem. 274, 30963–30968 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Martinez-Noel, G., Niedenthal, R., Tamura, T. & Harbers, K. A family of structurally related RING finger proteins interacts specifically with the ubiquitin-conjugating enzyme UbcM4. FEBS Lett. 454, 257–261 (1999).

    CAS  Article  Google Scholar 

  27. 27

    Lee, D.H. & Goldberg, A.L. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. 8, 397–403 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Gropper, R. et al. The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins. J. Biol. Chem. 266, 3602–3610 (1991).

    CAS  PubMed  Google Scholar 

  29. 29

    Barrett, A.J. et al. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem. J. 201, 189–198 (1982).

    CAS  Article  Google Scholar 

  30. 30

    Shimazu, R. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189, 1777–1782 (1999).

    CAS  Article  Google Scholar 

  31. 31

    da Silva, C.J., Soldau, K., Christen, U., Tobias, P.S. & Ulevitch, R.J. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2. J. Biol. Chem. 276, 21129–21135 (2001).

    Article  Google Scholar 

  32. 32

    Pugin, J., Ulevitch, R.J. & Tobias, P.S. A critical role for monocytes and CD14 in endotoxin-induced endothelial cell activation. J. Exp. Med. 178, 2193–2200 (1993).

    CAS  Article  Google Scholar 

  33. 33

    Sellati, T.J., Abrescia, L.D., Radolf, J.D. & Furie, M.B. Outer surface lipoproteins of Borrelia burgdorferi activate vascular endothelium in vitro. Infect. Immun. 64, 3180–3187 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Macfarlane, D.E. & Manzel, L. Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds. J. Immunol. 160, 1122–1131 (1998).

    CAS  PubMed  Google Scholar 

  35. 35

    Hoebe, K. et al. Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4, 1223–1229 (2003).

    CAS  Article  Google Scholar 

  36. 36

    Kitada, T. et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608 (1998).

    CAS  Article  Google Scholar 

  37. 37

    Aguilera, M., Oliveros, M., Martinez-Padron, M., Barbas, J.A. & Ferrus, A. Ariadne-1: a vital Drosophila gene is required in development and defines a new conserved family of ring-finger proteins. Genetics 155, 1231–1244 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Sakata, E. et al. Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain. EMBO Rep. 4, 301–306 (2003).

    CAS  Article  Google Scholar 

  39. 39

    Yamin, T.T. & Miller, D.K. The interleukin-1 receptor-associated kinase is degraded by proteasomes following its phosphorylation. J. Biol. Chem. 272, 21540–21547 (1997).

    CAS  Article  Google Scholar 

  40. 40

    Li, L., Cousart, S., Hu, J. & McCall, C.E. Characterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells. J. Biol. Chem. 275, 23340–23345 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Bosisio, D. et al. Stimulation of toll-like receptor 4 expression in human mononuclear phagocytes by interferon-γ: a molecular basis for priming and synergism with bacterial lipopolysaccharide. Blood 99, 3427–3431 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Marsik, C. et al. Endotoxaemia modulates Toll-like receptors on leucocytes in humans. Br. J. Haematol. 121, 653–656 (2003).

    CAS  Article  Google Scholar 

  43. 43

    Girnita, L., Girnita, A. & Larsson, O. Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor. Proc. Natl. Acad. Sci. USA 100, 8247–8252 (2003).

    CAS  Article  Google Scholar 

  44. 44

    Shenoy, S.K., McDonald, P.H., Kohout, T.A. & Lefkowitz, R.J. Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin. Science 294, 1307–1313 (2001).

    CAS  Article  Google Scholar 

  45. 45

    Cohen, B.D., Bariteau, J.T., Magenis, L.M. & Dias, J.A. Regulation of follitropin receptor cell surface residency by the ubiquitin-proteasome pathway. Endocrinology 144, 4393–4402 (2003).

    CAS  Article  Google Scholar 

  46. 46

    Yamakami, M., Yoshimori, T. & Yokosawa, H. Tom 1, a VHS domain-containing protein, interacts with Tollip, ubiquitin, and clathrin. J. Biol. Chem. 278, 52865–52872 (2003).

    CAS  Article  Google Scholar 

  47. 47

    Zhang, G. & Ghosh, S. Negative regulation of toll-like receptor-mediated signaling by Tollip. J. Biol. Chem. 277, 7059–7065 (2002).

    CAS  Article  Google Scholar 

  48. 48

    Chuang, T.H., Lee, J., Kline, L., Mathison, J.C. & Ulevitch, R.J. Toll-like receptor 9 mediates CpG-DNA signaling. J. Leukoc. Biol. 71, 538–544 (2002).

    CAS  PubMed  Google Scholar 

  49. 49

    Chuang, T.H. & Ulevitch, R.J. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur. Cytokine Netw. 11, 372–378 (2000).

    CAS  PubMed  Google Scholar 

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We thank J.F. Lo and W.Y. Hu for providing reagents and for discussions, and J.C. Mathison for comments on the manuscript. Supported by National Institutes of Health (AI15136, GM28485 and AI54523 to R.J.U.).

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Correspondence to Richard J Ulevitch.

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Chuang, T., Ulevitch, R. Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors. Nat Immunol 5, 495–502 (2004).

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