Deciphering non-canonical ubiquitin signaling: biology and methodology

Abstract

Ubiquitination is a dynamic post-translational modification that regulates virtually all cellular processes by modulating function, localization, interactions and turnover of thousands of substrates. Canonical ubiquitination involves the enzymatic cascade of E1, E2 and E3 enzymes that conjugate ubiquitin to lysine residues giving rise to monomeric ubiquitination and polymeric ubiquitination. Emerging research has established expansion of the ubiquitin code by non-canonical ubiquitination of N-termini and cysteine, serine and threonine residues. Generic methods for identifying ubiquitin substrates using mass spectrometry based proteomics often overlook non-canonical ubiquitinated substrates, suggesting that numerous undiscovered substrates of this modification exist. Moreover, there is a knowledge gap between in vitro studies and comprehensive understanding of the functional consequence of non-canonical ubiquitination in vivo. Here, we discuss the current knowledge about non-lysine ubiquitination, strategies to map the ubiquitinome and their applicability for studying non-canonical ubiquitination substrates and sites. Furthermore, we elucidate the available chemical biology toolbox and elaborate on missing links required to further unravel this less explored subsection of the ubiquitin system.

Keywords: E3 ligase; affinity purification; mass spectrometry; non-canonical ubiquitination; oxyester; proteomics; thioester; ubiquitin.

FIGURE 1

The ubiquitin cascade. (A) Ubiquitin is transferred to its substrates by a three-step enzymatic cascade involving E1 activating enzymes, E2 conjugating enzymes and E3 ligases. RING and U-box E3s coordinate the direct transfer of ubiquitin from E2 to the designated substrate, while transthiolating E3s covalently bind ubiquitin via their own catalytic cysteine. The final ubiquitination step occurs through a nucleophilic attack of the substrate on the electrophilic thioester-carbonyl of the E2-or E3-ubiquitin conjugate. (B) Depending on the substrate, these nucleophiles comprise lysine side chains, protein N-termini or side chains from unconventional amino acids such as cysteine, threonine or serine, thereby giving rise to isopeptide-, peptide-, thioester, or oxyester-linkages, respectively. (C) Phosphoribosyl-linked serine ubiquitination by Legionella pneumophila is mediated by the ADP ribosyl transferase (ART) and phosphodiesterase (PDE) domains of a single SidE family member. SidE^ART first ADP-ribosylates ubiquitin’s Arg42 through the transfer of ADPR from NAD⁺ to ubiquitin. Subsequently, the SidE PDE domains recognize this intermediate and catalyze the formation of a phosphodiester bond between ADP-ribosylated ubiquitin and a serine residue in the substrate protein.

FIGURE 2

Protein or peptide based enrichment strategies to identify the ubiquitinome by mass spectrometry analysis. (A) General workflow of mass spectrometry based proteomics experiment. Proteins are extracted from cells or tissue and digested into peptides using proteases. These peptides are separated by liquid-chromatography (LC) and analyzed by tandem mass spectrometry (MS/MS) to allow for protein identification based on fragmented ion spectra. Due to low abundance of ubiquitinated substrates in the total proteome, modified proteins are commonly enriched either on a protein- or peptide-based level. (B) Schematic overview of ubiquitin substrate enrichment strategies at a protein level. Exogenous expression or endogenous tagging of ubiquitin followed by purification of modified substrates from cell lysates using anti-epitope antibody conjugated beads (left). Pull-down of ubiquitinated proteins by immunoprecipitation using specific ubiquitin antibodies (middle). Epitope tagged ubiquitin-binding domains bind ubiquitin with high affinity and enrich substrates through pull-down with anti-epitope antibody conjugated beads (right). (C–E) Schematic overview of approaches to enrich ubiquitinated peptides following digestion. (C) Tryptic digestion of cell lysates leaves a GlyGly remnant at sites of ubiquitination. The diGly antibody recognizes this K-ε-GG epitope and enriches lysine-linked ubiquitinated peptides. (D) The UbiSite antibody binds the longer ubiquitin remnant following digestion with the protease Lys-C, enriching substrates regardless of the residue ubiquitin is conjugated to. Purified N-terminal-, cysteine-, serine-, threonine-, and lysine-linked ubiquitinated peptides are processed by trypsin to generate peptides of suitable length for mass spectrometry analysis. (E) The antibody-free approach for ubiquitination profiling (AFUP) blocks free ε-amine groups of lysine residues with formaldehyde, followed by cleavage of ubiquitin by deubiquitinating enzymes. The newly exposed ε-amine groups at lysine ubiquitin sites are labelled by NHS-SS-biotin and enriched with streptavidin beads.

FIGURE 3

Specific recognition of ubiquitin substrates by UbiSite antibody. Ubiquitin, Nedd8 and ISG15 are conjugated to substrates through their C-terminal glycine residue. DiGly antibodies bind to the GlyGly remnant left at the site of modification following trypsin digestion and thus recognize both ubiquitin as Nedd8 and ISG15 modified peptides. The UbiSite antibody recognizes the larger remnant left by Lys-C digestion, which is specific for ubiquitin.