Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W

Christopher W Davies,Carsten Schwerdtfeger,Ingrid E Wertz,Jawahar Sudhamsu,Andrey S Shaw,Simon E Vidal,Jennie R Lill,Christopher M Rose,Yi Jimmy Zeng,James T Koerber,Lilian Phu,Andrew S Peterson,Donald S Kirkpatrick,Trent B Hinkle,Frances-Rose Schumacher,Scott Chan Rosenberg

doi:10.1038/s41467-021-24669-6

Abstract

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.

Highlights

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the Ntermini of proteins, its substrate repertoire remains unclear
Using a combination of biochemical and structural methods, we show that these monoclonal antibody (mAb) predominately recognize the N-terminal diglycine with a relaxed selectivity for the third amino acid, enabling these mAbs to bind to a broad range of peptide sequences
As UBE2W is currently the only E2 known to mediate Nterminal ubiquitination of substrate proteins, we used an inducible UBE2W overexpression system and identified 73 putative UBE2W substrates, most of which are predicted to have disordered N-termini. Among these were two related deubiquitinases, UCHL1 and UCHL5, that were identified as targets of Nterminal ubiquitination; interestingly, we show that N-terminal ubiquitination is not a potent signal for degradation, but rather modulates deubiquitinase function

Summary

Introduction

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the Ntermini of proteins, 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. 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. These studies showed that engineered proteins lacking lysine residues or naturally occurring lysine-less proteins were still subject to proteasomal degradation, indirectly implicating N-terminal Ub as the degradation signal

Methods

Results

Conclusion