Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis [electronic resource]

• Ngôn ngữ: eng
• Ký hiệu phân loại: 579.2 *Viruses and subviral organisms
• Thông tin xuất bản: Washington, D.C. : Oak Ridge, Tenn. : United States. Dept. of Energy. Office of Science ; Distributed by the Office of Scientific and Technical Information, U.S. Dept. of Energy, 2015
• Mô tả vật lý: Size: p. 4029-4038 : , digital, PDF file.
• Bộ sưu tập: Metadata
• ID: 262084
• Chủ đề: 1. 59 basic biological sciences; 2. 60 applied life sciences; 3. Deubiquitinase; 4. Environmental molecular sciences laboratory; 5. Mass spectrometry; 6. Post-translational modification; 7. Substrate identification; 8. Ubiquitination

Tóm tắt

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.