How to Target Viral and Bacterial Effector Proteins Interfering with Ubiquitin Signaling

  • Gerbrand J. van der Heden van NoortEmail author
  • Huib Ovaa
Part of the Current Topics in Microbiology and Immunology book series (CT MICROBIOLOGY, volume 420)


Ubiquitination is a frequently occurring and very diverse posttranslational modification influencing a wide scope of cellular processes. Ubiquitin (Ub) has the unique ability to form eight different lysine-linked polymeric chains, mixed chains and engages with ubiquitin-like (Ubl) molecules. The distinct signals evoked by specific enzymes play a crucial role in, for instance, proteasome-mediated protein degradation, cell cycle regulation, and DNA damage responses. Due to the large variety of cellular functions that this posttranslational modification influences, the enzymes that construct such Ub modifications, and subsequently controle and degrade these signals, is enormous. In this chapter, we will discuss the current state-of-the-art of activity-based probes, reporter substrates, and other relevant tools based on Ub as recognition element, to study the enzymes involved in the complex system of ubiquitination.


  1. An H, Statsyuk AV (2013) Development of activity-based probes for ubiquitin and ubiquitin-like protein signaling pathways. J Am Chem Soc 135(45):16948–16962. Scholar
  2. An H, Statsyuk AV (2016) Facile synthesis of covalent probes to capture enzymatic intermediates during E1 enzyme catalysis. Chem Commun 52(12):2477–2480. Scholar
  3. Békés M, Rut W, Kasperkiewicz P, Mulder Monique PC, Ovaa H, Drag M, Lima Christopher D, Huang Tony T (2015) SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme. Biochem J 468(2):215–226. Scholar
  4. Bekes M, van Noort GJV, Ekkebus R, Ovaa H, Huang TT, Lima CD (2016) Recognition of Lys48-linked di-ubiquitin and deubiquitinating activities of the SARS coronavirus papain-like protease. Mol Cell 62(4):572–585. Scholar
  5. Bhogaraju S, Kalayil S, Liu Y, Bonn F, Colby T, Matic I, Dikic I (2016) Phosphoribosylation of ubiquitin promotes serine ubiquitination and impairs conventional ubiquitination. Cell 167(6):1636–1649.e1613. Scholar
  6. Borodovsky A, Kessler BM, Casagrande R, Overkleeft HS, Wilkinson KD, Ploegh HL (2001) A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J 20(18):5187–5196. Scholar
  7. Borodovsky A, Ovaa H, Kolli N, Gan-Erdene T, Wilkinson KD, Ploegh HL, Kessler BM (2002) Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem Biol 9(10):1149–1159. Scholar
  8. Buetow L, Huang DT (2016) Structural insights into the catalysis and regulation of E3 ubiquitin ligases. Nat Rev Mol Cell Biol 17:626. Scholar
  9. Dang LC, Melandri FD, Stein RL (1998) Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry 37(7):1868–1879. Scholar
  10. de Jong A, Merkx R, Berlin I, Rodenko B, Wijdeven RHM, El Atmioui D, Yalçin Z, Robson CN, Neefjes JJ, Ovaa H (2012) Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. ChemBioChem 13(15):2251–2258. Scholar
  11. de Jong A, Witting K, Kooij R, Flierman D, Ovaa H (2017) Release of enzymatically active deubiquitinating enzymes upon reversible capture by disulfide ubiquitin reagents. Angew Chem Int Ed 56(42):12967–12970. Scholar
  12. Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJR, Komander D, Ovaa H (2013) On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J Am Chem Soc 135(8):2867–2870. Scholar
  13. El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK, Ovaa H (2010) Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew Chem Int Ed 49(52):10149–10153. Scholar
  14. Faggiano S, Alfano C, Pastore A (2016) The missing links to link ubiquitin: methods for the enzymatic production of polyubiquitin chains. Anal Biochem 492:82–90. Scholar
  15. Flierman D, van der Heden van Noort GJ, Ekkebus R, Geurink PP, Mevissen TET, Hospenthal MK, Komander D, Ovaa H (2016) Non-hydrolyzable diubiquitin probes reveal linkage-specific reactivity of deubiquitylating enzymes mediated by S2 pockets. Cell Chem Biol 23(4):472–482. Scholar
  16. Geurink PP, El Oualid F, Jonker A, Hameed DS, Ovaa H (2012) A general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents. ChemBioChem 13(2):293–297. Scholar
  17. Geurink PP, van Tol BDM, van Dalen D, Brundel PJG, Mevissen TET, Pruneda JN, Elliott PR, van Tilburg GBA, Komander D, Ovaa H (2016) Development of diubiquitin-based FRET probes to quantify ubiquitin linkage specificity of deubiquitinating enzymes. ChemBioChem 17(9):816–820. Scholar
  18. Haj-Yahya N, Hemantha HP, Meledin R, Bondalapati S, Seenaiah M, Brik A (2014) Dehydroalanine-based diubiquitin activity probes. Org Lett 16(2):540–543. Scholar
  19. Hassiepen U, Eidhoff U, Meder G, Bulber J-F, Hein A, Bodendorf U, Lorthiois E, Martoglio B (2007) A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal Biochem 371(2):201–207. Scholar
  20. Kotewicz KM, Ramabhadran V, Sjoblom N, Vogel JP, Haenssler E, Zhang M, Behringer J, Scheck RA, Isberg RR (2017) A single Legionella effector catalyzes a multistep ubiquitination pathway to rearrange tubular endoplasmic reticulum for replication. Cell Host Microbe 21(2):169–181. Scholar
  21. Krist DT, Park S, Boneh GH, Rice SE, Statsyuk AV (2016) UbFluor: a mechanism-based probe for HECT E3 ligases. Chem Sci 7(8):5587–5595. Scholar
  22. Kumar KSA, Haj-Yahya M, Olschewski D, Lashuel HA, Brik A (2009) Highly efficient and chemoselective peptide ubiquitylation. Angew Chem Int Ed 48(43):8090–8094. Scholar
  23. Kumar KSA, Bavikar SN, Spasser L, Moyal T, Ohayon S, Brik A (2011) Total chemical synthesis of a 304 amino acid K48-linked tetraubiquitin protein. Angew Chem Int Ed 50(27):6137–6141. Scholar
  24. Lam YA, Xu W, DeMartino GN, Cohen RE (1997) Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385:737. Scholar
  25. Li G, Liang Q, Gong P, Tencer AH, Zhuang Z (2014) Activity-based diubiquitin probes for elucidating the linkage specificity of deubiquitinating enzymes. Chemical Commun (Camb) 50(2):216–218. Scholar
  26. Liu Q, Kistemaker HAV, Bhogaraju S, Dikic I, Overkleeft HS, van der Marel GA, Ovaa H, van der Heden van Noort GJ, Filippov DV (2018) A general approach towards triazole-linked adenosine diphosphate ribosylated peptides and proteins. Angew Chem Int Ed 57(6):1659–1662. Scholar
  27. Lu X, Olsen SK, Capili AD, Cisar JS, Lima CD, Tan DS (2010) Designed semisynthetic protein inhibitors of Ub/Ubl E1 activating enzymes. J Am Chem Soc 132(6):1748–1749. Scholar
  28. Madiraju C, Welsh K, Cuddy MP, Godoi PH, Pass I, Ngo T, Vasile S, Sergienko EA, Diaz P, Matsuzawa S-I, Reed JC (2012) TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors. J Biomol Screen 17(2):163–176. Scholar
  29. McGouran Joanna F, Gaertner Selina R, Altun M, Kramer Holger B, Kessler Benedikt M (2013) Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes. Chem Biol 20(12):1447–1455. Scholar
  30. Misra M, Kuhn M, Löbel M, An H, Statsyuk AV, Sotriffer C, Schindelin H (2017) Dissecting the specificity of adenosyl sulfamate inhibitors targeting the ubiquitin-activating enzyme. Structure 25(7):1120–1129.e1123. Scholar
  31. Moyal T, Bavikar SN, Karthikeyan SV, Hemantha HP, Brik A (2012) Polymerization behavior of a bifunctional ubiquitin monomer as a function of the nucleophile site and folding conditions. J Am Chem Soc 134(38):16085–16092. Scholar
  32. Mulder MPC, El Oualid F, ter Beek J, Ovaa H (2014) A native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study. ChemBioChem 15(7):946–949. Scholar
  33. Mulder MPC, Witting K, Berlin I, Pruneda JN, Wu K-P, Chang J-G, Merkx R, Bialas J, Groettrup M, Vertegaal ACO, Schulman BA, Komander D, Neefjes J, El Oualid F, Ovaa H (2016) A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. Nat Chem Biol 12(7):523–530. Scholar
  34. Mulder MPC, Merkx R, Witting KF, Hameed DS, El Atmioui D, Lelieveld L, Liebelt F, Neefjes J, Berlin I, Vertegaal ACO, Ovaa H (2018) Total chemical synthesis of SUMO and SUMO-based probes for profiling the activity of SUMO-specific proteases. Angew Chem Int Ed 57(29):8958–8962. Scholar
  35. Ohayon S, Spasser L, Aharoni A, Brik A (2012) Targeting deubiquitinases enabled by chemical synthesis of proteins. J Am Chem Soc 134(6):3281–3289. Scholar
  36. Orcutt SJ, Wu J, Eddins MJ, Leach CA, Strickler JE (2012) Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases. Biochim Biophys Acta (BBA) – Mol Cell Res 1823(11):2079–2086. Scholar
  37. Pao K-C, Stanley M, Han C, Lai Y-C, Murphy P, Balk K, Wood NT, Corti O, Corvol J-C, Muqit MMK, Virdee S (2016) Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation. Nat Chem Biol 12:324. Scholar
  38. Park S, Krist DT, Statsyuk AV (2015) Protein ubiquitination and formation of polyubiquitin chains without ATP, E1 and E2 enzymes. Chem Sci 6(3):1770–1779. Scholar
  39. Park S, Foote PK, Krist DT, Rice SE, Statsyuk AV (2017) UbMES and UbFluor: novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN. J Biol Chem 292(40):16539–16553. Scholar
  40. Pasunooti KK, Yang R, Vedachalam S, Gorityala BK, Liu C-F, Liu X-W (2009) Synthesis of 4-mercapto-l-lysine derivatives: potential building blocks for sequential native chemical ligation. Bioorg Med Chem Lett 19(22):6268–6271. Scholar
  41. Pickart CM, Rose IA (1986) Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin. J Biol Chem 261(22):10210–10217PubMedGoogle Scholar
  42. Pruneda Jonathan N, Durkin Charlotte H, Geurink Paul P, Ovaa H, Santhanam B, Holden David W, Komander D (2016) The molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases. Mol Cell 63(2):261–276. Scholar
  43. Puvar K, Zhou Y, Qiu J, Luo Z-Q, Wirth MJ, Das C (2017) Ubiquitin chains modified by the bacterial ligase SdeA are protected from deubiquitinase hydrolysis. Biochemistry. Scholar
  44. Qiu J, Sheedlo MJ, Yu K, Tan Y, Nakayasu ES, Das C, Liu X, Luo Z-Q (2016) Ubiquitination independent of E1 and E2 enzymes by bacterial effectors. Nature 533(7601):120–124. Scholar
  45. Qiu J, Yu K, Fei X, Liu Y, Nakayasu ES, Piehowski PD, Shaw JB, Puvar K, Das C, Liu X, Luo Z-Q (2017) A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination. Cell Res 27(7):865–881. Scholar
  46. Schneider T, Schneider D, Rösner D, Malhotra S, Mortensen F, Mayer TU, Scheffner M, Marx A (2014) Dissecting ubiquitin signaling with linkage-defined and protease resistant ubiquitin chains. Angew Chem Int Ed 53(47):12925–12929. Scholar
  47. Sheedlo MJ, Qiu J, Tan Y, Paul LN, Luo Z-Q, Das C (2015) Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination. Proc Natl Acad Sci 112(49):15090–15095. Scholar
  48. Spasser L, Brik A (2012) Chemistry and biology of the ubiquitin signal. Angew Chem Int Ed 51(28):6840–6862. Scholar
  49. Stanley M, Han C, Knebel A, Murphy P, Shpiro N, Virdee S (2015) Orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of E1 activating enzymes. ACS Chem Biol 10(6):1542–1554. Scholar
  50. Stewart MD, Ritterhoff T, Klevit RE, Brzovic PS (2016) E2 enzymes: more than just middle men. Cell Res 26:423. Scholar
  51. Tang S, Liang L-J, Si Y-Y, Gao S, Wang J-X, Liang J, Mei Z, Zheng J-S, Liu L (2017) Practical chemical synthesis of atypical ubiquitin chains by using an isopeptide-linked Ub isomer. Angew Chem Int Ed 56(43):13333–13337. Scholar
  52. Tirat A, Schilb A, Riou V, Leder L, Gerhartz B, Zimmermann J, Worpenberg S, Eidhoff U, Freuler F, Stettler T, Mayr L, Ottl J, Leuenberger B, Filipuzzi I (2005) Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2. Anal Biochem 343(2):244–255. Scholar
  53. Trang VH, Valkevich EM, Minami S, Chen Y-C, Ge Y, Strieter ER (2012) Nonenzymatic polymerization of ubiquitin: single-step synthesis and isolation of discrete ubiquitin oligomers. Angew Chem Int Ed 51(52):13085–13088. Scholar
  54. van der Heden van Noort GJ, Kooij R, Elliott PR, Komander D, Ovaa H (2017) Synthesis of poly-ubiquitin chains using a bifunctional ubiquitin monomer. Org Lett 19(24):6490–6493. Scholar
  55. van Tilburg GBA, Elhebieshy AF, Ovaa H (2016) Synthetic and semi-synthetic strategies to study ubiquitin signaling. Curr Opin Struct Biol 38:92–101. Scholar
  56. Ye Y, Blaser G, Horrocks MH, Ruedas-Rama MJ, Ibrahim S, Zhukov AA, Orte A, Klenerman D, Jackson SE, Komander D (2012) Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492(7428):266–270. Scholar
  57. Zhang X, Smits AH, van Tilburg GBA, Jansen PWTC, Makowski MM, Ovaa H, Vermeulen M (2017) An interaction landscape of ubiquitin signaling. Mol Cell 65(5):941–955.e948. Scholar
  58. Zhao X, Lutz J, Höllmüller E, Scheffner M, Marx A, Stengel F (2017) Identification of proteins interacting with ubiquitin chains. Angew Chem Int Ed 56(49):15764–15768. Scholar

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© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Gerbrand J. van der Heden van Noort
    • 1
    Email author
  • Huib Ovaa
    • 1
  1. 1.Department of Cell and Chemical Biology, Chemical ImmunologyOncode Institute, Leiden University Medical CentreLeidenThe Netherlands

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