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 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.
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Ernesto S
authors.initials: ES
authors.lastname: Nakayasu
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Sydor
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Roslyn N
authors.initials: RN
authors.lastname: Brown
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Sontag
authors.affiliation: National Center for Research in Energy and Materials, National Laboratory for Biosciences (LNBio) , Campinas, Sao Paulo 13083-970, Brazil.
authors.forename: Tiago J P
authors.initials: TJ
authors.lastname: Sobreira
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Gordon W
authors.initials: GW
authors.lastname: Slysz
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Humphrys
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Midwest Centre for Structural Genomics, University of Toronto , Toronto, Ontario M5G 1L6, Canada.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Skarina
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Midwest Centre for Structural Genomics, University of Toronto , Toronto, Ontario M5G 1L6, Canada.
authors.forename: Olena
authors.initials: O
authors.lastname: Onoprienko
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Midwest Centre for Structural Genomics, University of Toronto , Toronto, Ontario M5G 1L6, Canada.
authors.forename: Rosa
authors.initials: R
authors.lastname: Di Leo
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Brooke L
authors.initials: BL
authors.lastname: Deatherage Kaiser
authors.affiliation: Department of Molecular Microbiology & Immunology, Oregon Health & Science University , Portland, Oregon 97239, United States.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Charles
authors.initials: C
authors.lastname: Ansong
authors.affiliation: Department of Molecular Microbiology & Immunology, Oregon Health & Science University , Portland, Oregon 97239, United States.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Cambronne
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Midwest Centre for Structural Genomics, University of Toronto , Toronto, Ontario M5G 1L6, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.affiliation: Biological Science Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D007118:Immunoassay; D013058:Mass Spectrometry; D051379:Mice; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D012486:Salmonella typhimurium / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D001426:Bacterial Proteins; D064570:Ubiquitin-Specific Proteases
keywords: Ubiquitination; deubiquitinase; mass spectrometry; post-translational modification; substrate identification