Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W. © 2021. The Author(s).

Citation

Christopher W Davies, Simon E Vidal, Lilian Phu, Jawahar Sudhamsu, Trent B Hinkle, Scott Chan Rosenberg, Frances-Rose Schumacher, Yi Jimmy Zeng, Carsten Schwerdtfeger, Andrew S Peterson, Jennie R Lill, Christopher M Rose, Andrey S Shaw, Ingrid E Wertz, Donald S Kirkpatrick, James T Koerber. Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W. Nature communications. 2021 Jul 29;12(1):4608

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PMID: 34326324

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