Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W

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.

Current methodologies in protein ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

“…In addition, StUbEx PLUS (see below) is an antibody-free approach that is different from the above antibody-based approaches, showing no bias toward lysine and N-terminal ubiquitination. That's why only a small part of N-terminal ubiquitination sites identified by the anti-GGX mAbs-based approach were overlapped with the ubiquitination sites identified by UbiSite and StUbEx PLUS approaches [66,70,71]. This result nicely highlights the limitations and complementary of the current approaches.…”

“…However, current approaches, except the UbiSite approach, mainly focus on lysine ubiquitination profiling, there are limited approaches to analyzing the N-terminal ubiquitination, which hampers the functional study of protein N-terminal ubiquitination. To identify protein N-terminal ubiquitination, three approaches for profiling protein N-terminal ubiquitination, anti-GGX mAbs based approach, UbiSite based approach, and StUbEx PLUS approach, were reported [66,70,71]. For example, Davies et al generated four monoclonal antibodies (anti-GGX mAbs) that selectively recognize tryptic peptides with an N-terminal diGly remnant rather than the K-ε-GG group, realizing the specific enrichment and identification of protein N-terminal ubiquitination [70].…”

“…To identify protein N-terminal ubiquitination, three approaches for profiling protein N-terminal ubiquitination, anti-GGX mAbs based approach, UbiSite based approach, and StUbEx PLUS approach, were reported [66,70,71]. For example, Davies et al generated four monoclonal antibodies (anti-GGX mAbs) that selectively recognize tryptic peptides with an N-terminal diGly remnant rather than the K-ε-GG group, realizing the specific enrichment and identification of protein N-terminal ubiquitination [70]. UBE2W is the only E2 Ub conjugating enzyme reported to regulate the protein N-terminal ubiquitination.…”

Non-lysine ubiquitylation: Doing things differently

“…A major drawback of this technique however is that, due to the nature of the antibody epitope, it does not enrich for peptides originating from N-terminally ubiquitylated proteins. Recently other strategies, including the development of antibodies that recognize peptides bearing an N-terminal diglycine motif, have allowed detection of N-terminal ubiquitylation sites on a global scale ( Akimov et al, 2018a ; Akimov et al, 2018b ; Davies et al, 2021 ; Trulsson et al, 2022 ). These studies reveal that the relative abundance of N-terminal ubiquitin linkages is exceedingly low (to date several hundred sites have been identified), likely due to the fact that 80–90% of human proteins can be acetylated at their N-termini, a modification that would preclude N-terminal ubiquitylation ( Arnesen et al, 2009 ).…”

“…Thus far the ubiquitin conjugating enzyme UBE2W is the only E2 known to catalyze ubiquitylation of the N-termini of proteins ( Scaglione et al, 2013 ; Tatham et al, 2013 ; Davies et al, 2021 ). UBE2W strictly monoubiquitylates its substrates, priming them for polyubiquitylation by other E2/E3 complexes.…”