Advertisement

Ubiquitin-Activated Interaction Traps (UBAITs): Tools for Capturing Protein-Protein Interactions

  • Hazel F. O’Connor
  • Caleb D. Swaim
  • Larissa A. Canadeo
  • Jon M. HuibregtseEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)

Abstract

UBAITs (Ubiquitin-Activated Interaction Traps) are reagents that capitalize on the biochemistry of the ubiquitin system to covalently trap transient protein-protein interactions. UBAITs consist of an affinity-tagged protein of interest fused to a short linker followed by a C-terminal ubiquitin moiety. When charged in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety of the UBAIT has the potential to form an amide linkage with lysine side chains of a protein that interacts transiently with the protein of interest, thereby covalently trapping the protein-protein interaction. The partner protein can then be identified by affinity-based purification of the UBAIT coupled with mass spectroscopy methods. While originally designed to identify substrates of ubiquitin ligases, UBAITs can, in principle, be used for identifying interaction partners of virtually any protein of interest. Here we describe methods for utilizing UBAITs in both cell-based and in vitro experiments.

Key words

UBAIT Protein-protein interactions Ubiquitin ligases HECT E3 RING E3s 

References

  1. 1.
    O’Connor HF, Huibregtse JM (2017) Enzyme–substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases. Cell Mol Life Sci 74:3363–3375CrossRefGoogle Scholar
  2. 2.
    O’Leary CE, Lewis EL, Oliver PM (2015) Ubiquitylation as a rheostat for TCR signaling: from targeted approaches toward global profiling. Front Immunol 6:618CrossRefGoogle Scholar
  3. 3.
    Iconomou M, Saunders DN (2016) Systematic approaches to identify E3 ligase substrates. Biochem J 473:4083–4101.  https://doi.org/10.1042/BCJ20160719CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    O’Connor HF, Lyon N, Leung JW et al (2015) Ubiquitin-activated interaction traps (UBAITs) identify E3 ligase binding partners. EMBO Rep 16:1699–1712.  https://doi.org/10.15252/embr.201540620CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ling R, Colón E, Dahmus ME, Callis J (2000) Histidine-tagged ubiquitin substitutes for wild-type ubiquitin in Saccharomyces cerevisiae and facilitates isolation and identification of in vivo substrates of the ubiquitin pathway. Anal Biochem 282:54–64.  https://doi.org/10.1006/abio.2000.4586CrossRefPubMedGoogle Scholar
  6. 6.
    Peng J, Schwartz D, Elias JE et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21:921–926.  https://doi.org/10.1038/nbt849CrossRefPubMedGoogle Scholar
  7. 7.
    Swaim CD, Scott AF, Canadeo LA, Huibregtse JM (2017) Extracellular ISG15 signals cytokine secretion through the LFA-1 integrin receptor. Mol Cell 68:581–590.e5.  https://doi.org/10.1016/j.molcel.2017.10.003CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Hazel F. O’Connor
    • 1
  • Caleb D. Swaim
    • 1
  • Larissa A. Canadeo
    • 1
  • Jon M. Huibregtse
    • 1
    Email author
  1. 1.Department of Molecular BiosciencesUniversity of Texas at AustinAustinUSA

Personalised recommendations