Open AccessIdentification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis
Author(s) -
Ernesto Nakayasu,
M. Sydor,
Rebecca Brown,
Ryan Sontag,
Tiago J. P. Sobreira,
Gordon W. Slysz,
Daniel Humphrys,
T. Skarina,
Olena Onoprienko,
Rosa Di Leo,
Brooke L. Deatherage Kaiser,
Jie Li,
Charles Ansong,
Eric D. Cambronne,
Richard Smith,
Alexei Savchenko,
Joshua N. Adkins
Publication year - 2015
Publication title -
journal of proteome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.644
H-Index - 161
eISSN - 1535-3907
pISSN - 1535-3893
DOI - 10.1021/acs.jproteome.5b00574
Subject(s) - deubiquitinating enzyme , ubiquitin , biology , enzyme , salmonella , salmonella enterica , biochemistry , immunoprecipitation , computational biology , chemistry , bacteria , gene , genetics
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.