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Title: Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis

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 model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous 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. Finally, 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.

Authors:
 [1];  [1];  [1];  [1];  [2];  [1];  [1];  [3];  [3];  [3];  [1];  [4];  [1];  [4];  [1];  [3];  [1]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Science Division
  2. Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, Sao Paulo. National Lab. for Biosciences (LNBio)
  3. Univ. of Toronto, ON (Canada). Midwest Centre for Structural Genomics, Dept. of Chemical Engineering and Applied Chemistry
  4. Oregon Health & Science Univ., Portland, OR (United States), Dept. of Molecular Microbiology & Immunology
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH)
OSTI Identifier:
1243259
Report Number(s):
PNNL-SA-101042
Journal ID: ISSN 1535-3893; 47623; 400412000
Grant/Contract Number:  
AC05-76RL01830; GM094585; GM094623
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Proteome Research
Additional Journal Information:
Journal Volume: 14; Journal Issue: 9; Journal ID: ISSN 1535-3893
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ubiquitination; deubiquitinase; post-translational modification; substrate identification; mass spectrometry; Environmental Molecular Sciences Laboratory

Citation Formats

Nakayasu, Ernesto S., Sydor, Michael A., Brown, Roslyn N., Sontag, Ryan L., Sobreira, Tiago, Slysz, Gordon W., Humphrys, Daniel R., Skarina, Tatiana, Onoprienko, Olena, Di Leo, Rosa, Kaiser, Brooke L. Deatherage, Li, Jie, Ansong, Charles, Cambronne, Eric, Smith, Richard D., Savchenko, Alexei, and Adkins, Joshua N. Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis. United States: N. p., 2015. Web. doi:10.1021/acs.jproteome.5b00574.
Nakayasu, Ernesto S., Sydor, Michael A., Brown, Roslyn N., Sontag, Ryan L., Sobreira, Tiago, Slysz, Gordon W., Humphrys, Daniel R., Skarina, Tatiana, Onoprienko, Olena, Di Leo, Rosa, Kaiser, Brooke L. Deatherage, Li, Jie, Ansong, Charles, Cambronne, Eric, Smith, Richard D., Savchenko, Alexei, & Adkins, Joshua N. Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis. United States. doi:10.1021/acs.jproteome.5b00574.
Nakayasu, Ernesto S., Sydor, Michael A., Brown, Roslyn N., Sontag, Ryan L., Sobreira, Tiago, Slysz, Gordon W., Humphrys, Daniel R., Skarina, Tatiana, Onoprienko, Olena, Di Leo, Rosa, Kaiser, Brooke L. Deatherage, Li, Jie, Ansong, Charles, Cambronne, Eric, Smith, Richard D., Savchenko, Alexei, and Adkins, Joshua N. Mon . "Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis". United States. doi:10.1021/acs.jproteome.5b00574. https://www.osti.gov/servlets/purl/1243259.
@article{osti_1243259,
title = {Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis},
author = {Nakayasu, Ernesto S. and Sydor, Michael A. and Brown, Roslyn N. and Sontag, Ryan L. and Sobreira, Tiago and Slysz, Gordon W. and Humphrys, Daniel R. and Skarina, Tatiana and Onoprienko, Olena and Di Leo, Rosa and Kaiser, Brooke L. Deatherage and Li, Jie and Ansong, Charles and Cambronne, Eric and Smith, Richard D. and Savchenko, Alexei and Adkins, Joshua N.},
abstractNote = {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 model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous 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. Finally, 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.},
doi = {10.1021/acs.jproteome.5b00574},
journal = {Journal of Proteome Research},
number = 9,
volume = 14,
place = {United States},
year = {2015},
month = {7}
}

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