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Abstract
Ubiquitin is a small (76 amino acid) evolutionarily conserved protein that is covalently conjugated to lysine residues (or rarely N-terminal amines) on substrate polypeptides, to alter their half-life, localization or function. Ubiquitin itself also contains seven internal lysine residues, all of which can be modified by additional ubiquitin molecules to form extended ubiquitin “chains”. Monoubiquitylation or different types of ubiquitin chain linkages can confer very different biological outcomes to a protein target.
The ubiquitin conjugation process is carried out via a three-step enzymatic cascade consisting of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). More than 600 genes coding for predicted ubiquitin E3 ligases have been identified in the human genome, along with just 35 active E2 genes and two E1s. Importantly, the specific subset of E3s that interacts with each E2, and the specific biological functions of most of the E2 enzymes, have not been elucidated.
To better understand E2 functions, I conducted a series of interactome and gene essentiality screens. Proximity-dependent biotinylation (BioID) was performed in Flp-In T-REx 293 cells to identify interacting partners for 31 different E2 proteins. 2296 high confidence protein-protein interactions were identified amongst 964 different polypeptides. To identify E2 genes (and other genes linked to protein homeostasis) essential for cell fitness, CRISPR/Cas9 dropout screens were also performed in HEK293 cells, along with two different lung cancer lines, NCI-H1975 and SW1573. Several different context-specific essential E2 genes (and many other genes linked to protein homeostasis) were identified in this screen, indicating that E2s may represent viable therapeutic targets. Finally, two common methods have been used to identify the types of ubiquitin linkages that can be generated by specific E2-E3 pairs: in vitro autoubiquitylation reactions using recombinant ubiquitin proteins bearing a single lysine-to-arginine mutation, or direct identification using mass spectrometry. We compared these two methods by characterizing the linkage specificity of 12 different E2-E3 combinations, and demonstrated that mass spectrometry-based methods yield far superior results. Together, my work has thus provided important biological context to a poorly understood component of the ubiquitin system, the E2 family.
