Site-Specific Nanobody Inhibitors of the Proteasomal Deubiquitinase UCH37

Abstract

Dysregulation of the ubiquitin (Ub) proteasome system (UPS) is linked to numerous human diseases, making its components attractive therapeutic targets. Among these, the proteasomal deubiquitinase UCH37 (also known as UCHL5) has two distinct Ub-processing activities: C-terminal Ub hydrolysis and K48-linkage-specific chain debranching. Separating these activities has not been possible with existing small-molecule inhibitors, which target the catalytic site and broadly affect both functions along with many other DUBs. Here, we report the development of two site-specific nanobody inhibitors of UCH37 that together enable this separation. Using yeast surface display, we generated Nb-cS1, which binds the canonical Ub-binding (S1) site and blocks both activities, and Nb-ncS1, which binds the non-canonical K48-specific (ncS1) site and selectively impairs chain debranching without affecting C-terminal Ub hydrolysis. X-ray crystallography and hydrogen-deuterium exchange mass spectrometry define the distinct binding surfaces engaged by each nanobody. In cells, both nanobodies selectively engage UCH37, with Nb-ncS1 showing the cleanest target engagement. These tools provide the first means to independently interrogate UCH37s two enzymatic activities and offer new approaches for dissecting the distinct roles of UCH37 in proteasomal and chromatin-associated contexts.