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.
Institute of Biochemistry II, Goethe University Frankfurt-Medical Faculty, Frankfurt, Germany.
Katarzyna
K
Kliza
Proteome Center Tuebingen, Interfaculty Institute of Cell Biology, University of Tuebingen, Tuebingen, Germany.
Christoph
C
Taumer
Department of Chemistry and Biochemistry, Queen Mary University of London, London, UK.
Irene
I
Pinzuti
Proteome Center Tuebingen, Interfaculty Institute of Cell Biology, University of Tuebingen, Tuebingen, Germany.
Mirita
M
Franz-Wachtel
Structural Biology Science Technology Platform, Francis Crick Institute, London, UK.
Simone
S
Kunzelmann
Department of Chemistry and Biochemistry, Queen Mary University of London, London, UK.
Benjamin
http://orcid.org/0000-0003-3634-6997
B
Stieglitz
Proteome Center Tuebingen, Interfaculty Institute of Cell Biology, University of Tuebingen, Tuebingen, Germany.
Boris
B
Macek
Institute of Biochemistry II, Goethe University Frankfurt-Medical Faculty, Frankfurt, Germany.
Koraljka
K
Husnjak
D010450:Endopeptidases / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B / Q000378:metabolism; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D048029:TNF Receptor-Associated Factor 6; C000604754:Tifab protein, human; D014409:Tumor Necrosis Factor-alpha; D025821:Polyubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C582107:OTULIN protein, human