Site-Specific Nanobody Inhibitors of the Proteasomal Deubiquitinase UCH37

Dysregulation of the ubiquitin (Ub) proteasome system (UPS) is linked to numerous human diseases, making its components, particularly deubiquitinases (DUBs), attractive therapeutic targets. UCH37 (also known as UCHL5), a proteasomal DUB, plays roles in protein degradation, DNA repair, and transcription and is overexpressed in several cancers. Despite its relevance, the precise functions of UCH37 within the proteasome and the INO80 chromatin remodeling complex remain unclear. Current small-molecule inhibitors exhibit broad, off-target effects, limiting their utility both therapeutically and as probes for UCH37 function. Here, we report the development of highly potent, selective nanobody-based inhibitors for UCH37. Using yeast surface display, we generated nanobodies targeting distinct Ub-binding sites on UCH37, with binding specificity confirmed by X-ray crystallography and hydrogen-deuterium exchange mass spectrometry. We find that the nanobody targeting the canonical Ub binding site serves as a pan-UCH37 inhibitor, while the one binding to the K48 chain-specific site selectively inhibits Ub chain debranching. Expression of this Ub chain debranching-specific nanobody in cells enabled identification of proteins whose degradation depends on UCH37. This work provides novel tools for investigating the biological functions of UCH37 and lays the groundwork for evaluating its potential as a therapeutic target.