Identification and Validation of ISG15 Target Proteins

Part of the Subcellular Biochemistry book series (SCBI, volume 54)


ISG15 is an interferon-induced ubiquitin-like protein (Ubl) that has antiviral properties. The core E1, E2 and E3 enzymes for conjugation of human ISG15 are Ube1L, UbcH8 and Herc5, all of which are induced at the transcriptional level by Type 1 interferon signaling. Several proteomics studies have, together, identified over 300 cellular proteins as ISG15 targets. These targets include a broad range of constitutively expressed proteins and approximately 15 interferon-induced proteins. This chapter provides an overview of the target identification process and the validation of these targets. We also discuss the limited number of examples where the biochemical effect of ISG15 conjugation on target proteins has been characterized.


Human Physiology Identification Process Target Protein Cellular Protein Transcriptional Level 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Seth RB, Sun L, Chen ZJ. Antiviral innate immunity pathways. Cell Res 2006; 16:141–7.PubMedCrossRefGoogle Scholar
  2. 2.
    Trinchieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 2007; 7:179–90.PubMedCrossRefGoogle Scholar
  3. 3.
    Farrell PJ, Broeze RJ, Lengyel P. Accumulation of an mRNA and protein in interferon-treated Ehrlich ascites tumour cells. Nature 1979; 279:523–5.PubMedCrossRefGoogle Scholar
  4. 4.
    Haas AL, Ahrens P, Bright PM et al. Interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin. J Biol Chem 1987; 262:11315–23.PubMedGoogle Scholar
  5. 5.
    Loeb KR, Haas AL. The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins. J Biol Chem 1992; 267:7806–13.PubMedGoogle Scholar
  6. 6.
    Lenschow DJ, Giannakopoulos NV, Gunn LJ et al. Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo. J Virol 2005; 79:13974–83.PubMedCrossRefGoogle Scholar
  7. 7.
    Okumura A, Pitha PM, Harty RN. ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc Natl Acad Sci USA 2008; 105:3974–9.PubMedCrossRefGoogle Scholar
  8. 8.
    Okumura A, Lu G, Pitha-Rowe I et al. Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15. Proc Natl Acad Sci USA 2006; 103:1440–5.PubMedCrossRefGoogle Scholar
  9. 9.
    Hsiang TY, Zhao C, Krug RM. Interferon-induced ISG15 conjugation inhibits influenza A virus gene expression and replication in human cells. J Virol 2009; 83:5971–7.PubMedCrossRefGoogle Scholar
  10. 10.
    Ritchie KJ, Malakhov MP, Hetherington CJ et al. Dysregulation of protein modification by ISG15 results in brain cell injury. Genes Dev 2002; 16:2207–12.PubMedCrossRefGoogle Scholar
  11. 11.
    Malakhov MP, Malakhova OA, Kim KI et al. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J Biol Chem 2002; 277:9976–81.PubMedCrossRefGoogle Scholar
  12. 12.
    Liu LQ, Ilaria R Jr, Kingsley PD et al. A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ET O-expressing mice, functions in hematopoietic cell differentiation. Mol Cell Biol 1999; 19:3029–38.PubMedGoogle Scholar
  13. 13.
    Knobeloch KP, Utermohlen O, Kisser A et al. Reexamination of the role of ubiquitin-like modifier ISG15 in the phenotype of UBP43-deficient mice. Mol Cell Biol 2005; 25:11030–4.PubMedCrossRefGoogle Scholar
  14. 14.
    Catic A, Fiebiger E, Korbel GA et al. Screen for ISG15-crossreactive deubiquitinases. PLoS ONE 2007; 2:e679.PubMedCrossRefGoogle Scholar
  15. 15.
    Dastur A, Beaudenon S, Kelley M et al. Herc5, an interferon-induced HECT E3 enzyme, is required for conjugation of ISG15 in human cells. J Biol Chem 2006; 281:4334–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Yuan W, Krug RM. Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. EMBO J 2001; 20:362–71.PubMedCrossRefGoogle Scholar
  17. 17.
    Durfee LA, Kelley ML, Huibregtse JM. The basis for selective E1-E 2 interactions in the ISG15 conjugation system. J Biol Chem 2008; 283:23895–902.PubMedCrossRefGoogle Scholar
  18. 18.
    Kim KI, Giannakopoulos NV, Virgin HW et al. Interferon-Inducible Ubiquitin E2, Ubc8, Is a Conjugating Enzyme for Protein ISGylation. Mol Cell Biol 2004; 24:9592–600.PubMedCrossRefGoogle Scholar
  19. 19.
    Zhao C, Beaudenon SL, Kelley ML et al. The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-alpha/beta-induced ubiquitin-like protein. Proc Natl Acad Sci USA 2004; 101:7578–82.PubMedCrossRefGoogle Scholar
  20. 20.
    Chin LS, Vavalle JP, Li L. Staring, a novel E3 ubiquitin-protein ligase that targets syntaxin 1 for degradation. J Biol Chem 2002; 277:35071–9.PubMedCrossRefGoogle Scholar
  21. 21.
    Kumar S, Kao WH, Howley PM. Physical interaction between specific E2 and Hect E3 enzymes determines functional cooperativity. J Biol Chem 1997; 272:13548–54.PubMedCrossRefGoogle Scholar
  22. 22.
    Moynihan TP, Ardley HC, Nuber U 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 1999; 274:30963–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Niwa J, Ishigaki S, Doyu M et al. A novel centrosomal ring-finger protein, dorfin, mediates ubiquitin ligase activity. Biochem Biophys Res Commun 2001; 281:706–13.PubMedCrossRefGoogle Scholar
  24. 24.
    Tanaka K, Suzuki T, Chiba T et al. Parkin is linked to the ubiquitin pathway. J Mol Med 2001; 79:482–94.PubMedCrossRefGoogle Scholar
  25. 25.
    Urano T, Saito T, Tsukui T et al. Efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth. Nature 2002; 417:871–5.PubMedCrossRefGoogle Scholar
  26. 26.
    Zhang Y, Gao J, Chung KK 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 2000; 97:13354–9.PubMedCrossRefGoogle Scholar
  27. 27.
    Wong JJ, Pung YF, Sze NS et al. HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets. Proc Natl Acad Sci USA 2006; 103:10735–40.PubMedCrossRefGoogle Scholar
  28. 28.
    Takeuchi T, Inoue S, Yokosawa H. Identification and Herc5-mediated ISGylation of novel target proteins. Biochem Biophys Res Commun 2006; 348:473–7.PubMedCrossRefGoogle Scholar
  29. 29.
    Hochrainer K, Mayer H, Baranyi U et al. The human HERC family of ubiquitin ligases: novel members, genomic organization, expression profiling and evolutionary aspects. Genomics 2005; 85:153–64.PubMedCrossRefGoogle Scholar
  30. 30.
    Chang YG, Yan XZ, Xie YY et al. Different roles for two ubiquitin-like domains of ISG15 in protein modification. J Biol Chem 2008; 283:13370–7.PubMedCrossRefGoogle Scholar
  31. 31.
    Lenschow DJ, Lai C, Frias-Staheli N et al. IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes and Sindbis viruses. Proc Natl Acad Sci USA 2007; 104:1371–6.PubMedCrossRefGoogle Scholar
  32. 32.
    Lai C, Struckhoff JJ, Schneider J et al. Mice lacking the ISG15 E1 enzyme UbE1L demonstrate increased susceptibility to both mouse-adapted and nonmouse-adapted influenza B virus infection. J Virol 2009; 83:1147–51.PubMedCrossRefGoogle Scholar
  33. 33.
    Giannakopoulos NV, Arutyunova E, Lai C et al. ISG15 Arg151 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of Sindbis virus. J Virol 2009; 83:1602–10.PubMedCrossRefGoogle Scholar
  34. 34.
    Lindner HA, Lytvyn V, Qi H et al. Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease. Arch Biochem Biophys 2007; 466:8–14.PubMedCrossRefGoogle Scholar
  35. 35.
    Guerra S, Caceres A, Knobeloch KP et al. Vaccinia virus E3 protein prevents the antiviral action of ISG15. PLoS Pathog 2008; 4:e1000096.PubMedCrossRefGoogle Scholar
  36. 36.
    Peng J, Schwartz D, Elias JE et al. A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 2003; 21:921–6.PubMedCrossRefGoogle Scholar
  37. 37.
    Kirkpatrick DS, Denison C, Gygi SP. Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat Cell Biol 2005; 7:750–7.PubMedCrossRefGoogle Scholar
  38. 38.
    Denison C, Kirkpatrick DS, Gygi SP. Proteomic insights into ubiquitin and ubiquitin-like proteins. Curr Opin Chem Biol 2005; 9:69–75.PubMedCrossRefGoogle Scholar
  39. 39.
    Giannakopoulos NV, Luo JK, Papov V et al. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochem Biophys Res Commun 2005; 336:496–506.PubMedCrossRefGoogle Scholar
  40. 40.
    Zhao C, Denison C, Huibregtse JM et al. Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways. Proc Natl Acad Sci USA 2005; 102:10200–5.PubMedCrossRefGoogle Scholar
  41. 41.
    Panse VG, Hardeland U, Werner T et al. A proteome-wide approach identifies sumoylated substrate proteins in yeast. J Biol Chem 2004; 279:41346–51.Google Scholar
  42. 42.
    Denison C, Rudner AD, Gerber SA et al. A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol Cell Proteomics 2005; 4:246–54.PubMedCrossRefGoogle Scholar
  43. 43.
    Sen GC, Sarkar SN. The interferon-stimulated genes: targets of direct signaling by interferons, double-stranded RNA and viruses. Curr Top Microbiol Immunol 2007; 316:233–50.PubMedCrossRefGoogle Scholar
  44. 44.
    Sen GC. Novel functions of interferon-induced proteins. Semin Cancer Biol 2000; 10:93–101.PubMedCrossRefGoogle Scholar
  45. 45.
    Zou W, Papov V, Malakhova O et al. ISG15 modification of ubiquitin E2 Ubc13 disrupts its ability to form thioester bond with ubiquitin. Biochem Biophys Res Commun 2005; 336:61–8.PubMedCrossRefGoogle Scholar
  46. 46.
    Takeuchi T, Yokosawa H. ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity. Biochem Biophys Res Commun 2005; 336:9–13.PubMedCrossRefGoogle Scholar
  47. 47.
    Minakawa M, Sone T, Takeuchi T et al. Regulation of the nuclear factor (NF)-kappaB pathway by ISGylation. Biol Pharm Bull 2008; 31:2223–7.PubMedCrossRefGoogle Scholar
  48. 48.
    Stossel TP, Condeelis J, Cooley L et al. Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol 2001; 2:138–45.PubMedCrossRefGoogle Scholar
  49. 49.
    Jeon YJ, Choi JS, Lee JY et al. Filamin B serves as a molecular scaffold for type I interferon-induced c-Jun NH2-terminal kinase signaling pathway. Mol Biol Cell 2008; 19:5116–30.PubMedCrossRefGoogle Scholar
  50. 50.
    Takeuchi T, Kobayashi T, Tamura S et al. Negative regulation of protein phosphatase 2Cbeta by ISG15 conjugation. FEBS Lett 2006; 580:4521–6.PubMedCrossRefGoogle Scholar
  51. 51.
    Okumura F, Zou W, Zhang DE. ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP. Genes Dev 2007; 21:255–60.PubMedCrossRefGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2010

Authors and Affiliations

  1. 1.Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular BiologyThe University of Texas at AustinAustinUSA

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