Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.
Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current ... methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-REx HEK293T cells and validated several newly identified linear polyubiquitin targets. We demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for NFκB signaling.
Mesh Terms:
Endopeptidases, HEK293 Cells, HeLa Cells, Humans, NF-kappa B, Polyubiquitin, Protein Processing, Post-Translational, Signal Transduction, TNF Receptor-Associated Factor 6, Tumor Necrosis Factor-alpha, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitination
Endopeptidases, HEK293 Cells, HeLa Cells, Humans, NF-kappa B, Polyubiquitin, Protein Processing, Post-Translational, Signal Transduction, TNF Receptor-Associated Factor 6, Tumor Necrosis Factor-alpha, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitination
Nat. Methods
Date: May. 01, 2017
PubMed ID: 28319114
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