Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Science Division
- Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, Sao Paulo. National Lab. for Biosciences (LNBio)
- Univ. of Toronto, ON (Canada). Midwest Centre for Structural Genomics, Dept. of Chemical Engineering and Applied Chemistry
- Oregon Health & Science Univ., Portland, OR (United States), Dept. of Molecular Microbiology & Immunology
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.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
- Grant/Contract Number:
- AC05-76RL01830; GM094585; GM094623
- OSTI ID:
- 1243259
- Report Number(s):
- PNNL-SA-101042; 47623; 400412000
- Journal Information:
- Journal of Proteome Research, Vol. 14, Issue 9; ISSN 1535-3893
- Publisher:
- American Chemical Society (ACS)Copyright Statement
- Country of Publication:
- United States
- Language:
- English
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journal | July 2016 |
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