A novel antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W

ABSTRACT: Protein ubiquitination plays an essential role in cellular physiology. Contrary to canonical ubiquitination at internal lysine residues, the physiological functions of N-terminal ubiquitination remain enigmatic. To identify endogenous N-terminally-ubiquitinated substrates, we discovered four novel 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 solved the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. These antibodies were used in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates included UCHL1 and UCHL5, where N-terminal ubiquitination distinctly altered deubiquitinase (DUB) activity. This new antibody toolkit for global profiling of endogenous N-terminal ubiquitination sites will enable future work to decipher the physiological functions of N-terminal ubiquitination.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: JT Koerber  

PROVIDER: MSV000086537 | MassIVE | Mon Nov 30 18:02:00 GMT 2020

REPOSITORIES: MassIVE