Identification of salmonella typhimurium deubiquitinase ssel substrates by immunoaffinity enrichment and quantitative proteomic analysis

Ernesto S. Nakayasu, Michael A. Sydor, Roslyn N. Brown, Ryan L. Sontag, Tiago J.P. Sobreira, Gordon W. Slysz, Daniel R. Humphrys, Tatiana Skarina, Olena Onoprienko, Rosa Di Leo, Brooke L. Deatherage Kaiser, Jie Li, Charles Ansong, Eric D. Cambronne, Richard D. Smith, Alexei Savchenko, Joshua N. Adkins

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here, we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification, and global quantitative proteomic analysis. As a model system to identify substrates, we used a virulence-related deubiquitinase, SseL, secreted by Salmonella enterica serovar Typhimurium into host cells. Using this approach, two SseL substrates were identified in the RAW 264.7 murine macrophage-like cell line, S100A6 and heterogeneous nuclear ribonuclear protein K, in addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. This method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.

Original languageEnglish (US)
Pages (from-to)4029-4038
Number of pages10
JournalJournal of Proteome Research
Volume14
Issue number9
DOIs
StatePublished - Sep 4 2015

Keywords

  • Ubiquitination
  • deubiquitinase
  • mass spectrometry
  • post-translational modification
  • substrate identification

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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    Nakayasu, E. S., Sydor, M. A., Brown, R. N., Sontag, R. L., Sobreira, T. J. P., Slysz, G. W., Humphrys, D. R., Skarina, T., Onoprienko, O., Di Leo, R., Deatherage Kaiser, B. L., Li, J., Ansong, C., Cambronne, E. D., Smith, R. D., Savchenko, A., & Adkins, J. N. (2015). Identification of salmonella typhimurium deubiquitinase ssel substrates by immunoaffinity enrichment and quantitative proteomic analysis. Journal of Proteome Research, 14(9), 4029-4038. https://doi.org/10.1021/acs.jproteome.5b00574