When what's at the beginning matters:decoding… (F32GM166919) – Project Grant to University Of Washington

Abstract: Ubiquitination is a protein modification required for the regulation of protein stability, structure, function, and localization in cells. Ubiquitination's importance is underscored by successful targeting of the ubiquitin system in novel therapeutics for myeloid leukemia and other blood cancers, as well as its implication in other cancers and neurodegenerative diseases that still have unmet clinical needs. Careful coordination of the E1, E2, and E3 enzymes that install this mark and deubiquitinases (dubs) that remove IT enables proper cell function, yet there are persistent knowledge gaps in the mechanism and regulation of the enzymes involved. One such GAP is the mechanism and role of UBE2W, an E2 implicated in neurodegeneration and breast cancer and the only known E2 enzyme responsible for n-terminal ubiquitination of proteins (instead of common lysine modification). UBE2W's activity relies on a disordered c-terminal extension to recognize its n-terminally disordered substrates, nucleating broader questions to how a disordered protein can recognize another, as well as how this confers specificity for protein n-termini over lysine sidechains. UBE2W also contains a unique HPH motif, a variation on a conserved HPN motif in other E2S, whose role is also unclear. In this proposal, the goal is to fill knowledge gaps in UBE2W's mechanism through completion of aim 1: elucidate structural and functional roles of unique features of UBE2W. To do this, site-directed mutagenesis and genetic code expansion will be done to parse out the roles of UBE2W's HPH motif and residues in UBE2W's c-terminus in substrate recognition and catalysis. UBE2W mutants will be screened for changes in activity by in vitro ubiquitylation assays and structural changes by NMR spectroscopy, revealing the roles of residues in ubiquitin binding, substrate interaction, and catalysis. Beyond questions of its mechanism, the biological roles of UBE2W are still unknown, largely due to a lack of tools to identify its substrates. A recent study identified two dubs, UCHL1 and UCHL5, as substrates of UBE2W and demonstrated that n-terminal ubiquitination differentially altered their activities, suggesting a broader regulatory role of UBE2W in modulating ubiquitin removal. The mechanism and downstream biological effects of this regulation remain unexplored, so these knowledge gaps will be filled through the completion of aim 2: evaluate the impact of n-terminal ubiquitination on DUB structure and function and aim 3: investigate the role of UBE2W in regulating DUB enzymes in vivo. In aim 2, NMR spectroscopy and functional activity assays will be done to determine the allosteric effects that drive differences in DUB structure and activity upon n-terminal ubiquitination. To determine the role of UBE2W's regulation of DUB enzymes in vivo in aim 3, UBE2W knockout experiments will be performed in the nematode worm c. Elegans to determine the downstream effects of UBE2W-mediated DUB regulation in the health and stress response of the worms. Completion of these aims will shed light on both UBE2W's mechanism and biological roles, creating a more complete view of n-terminal ubiquitination and regulation in human health and disease.

Action	Obligated	Date
Amendment	$76,300	Aug 20, 2026
Original award	$0	Aug 20, 2026

When what's at the beginning matters:decoding how UBE2W shapes protein function

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