Identification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis

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 base...

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Bibliographic Details
Published in:Journal of proteome research 2015-09, Vol.14 (9), p.4029-4038
Main Authors: Nakayasu, Ernesto S, Sydor, Michael A, Brown, Roslyn N, Sontag, Ryan L, Sobreira, Tiago J. P, Slysz, Gordon W, Humphrys, Daniel R, Skarina, Tatiana, Onoprienko, Olena, Di Leo, Rosa, Deatherage Kaiser, Brooke L, Li, Jie, Ansong, Charles, Cambronne, Eric D, Smith, Richard D, Savchenko, Alexei, Adkins, Joshua N
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
Animals
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
BASIC BIOLOGICAL SCIENCES
Cell Line
cell lines
deubiquitinase
Environmental Molecular Sciences Laboratory
Immunoassay
Mass Spectrometry
Mice
post-translational modification
Protein Interaction Mapping
Protein Processing, Post-Translational
proteome
proteomics
Proteomics - methods
Salmonella Typhimurium
Salmonella typhimurium - metabolism
substrate identification
ubiquitin
ubiquitin-protein ligase
Ubiquitin-Specific Proteases - chemistry
Ubiquitin-Specific Proteases - metabolism
Ubiquitination
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Summary: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.
ISSN:1535-3893
1535-3907
1535-3907
DOI:10.1021/acs.jproteome.5b00574