The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

Spreading and migration of the basal cells neighboring a wound is essential for airway epithelial repair. To gain insight into the molecular mechanisms that govern these cellular processes, we asked whether normal human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls migration and invasion. Herein, we report that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C activator, induced reorganization of cytoskeletal structure in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. Z-stack scanning confocal microscopy showed that PDBu-induced podosome-like structures contain actin-rich columns that arise from the ventral surface of the cell, and also revealed the presence of circular ruffles/waves at the dorsal cell surface. The molecular components of these cytoskeletal structures were determined with immunofluorescent staining. Using in situ zymography, we demonstrated that PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased epithelial cell invasion across Transwell chamber. Podosomes and circular dorsal ruffles may be important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration.

A bipolar mitotic spindle facilitates the equal segregation of chromosomes to two daughter cells. To achieve bipolar attachment of microtubules to kinetochores of sister chromatids, chromatids must remain paired after replication. This cohesion is mediated by the conserved cohesin complex comprised of SMC1, SMC3, SCC1, and either SA1 or SA2 in humans. Because defects in spindle assembly or sister chromatid cohesion can lead to aneuploidy in daughter cells, proper regulation of these processes is essential for fidelity in chromosome segregation. In an RNAi screen for regulators of spindle assembly, we identify the deubiquitinase USP37 as a regulator of mitotic progression, centrosome integrity, and chromosome alignment. USP37 associates with cohesin and contributes to sister chromatid resolution. Cohesion defects are rescued by expression of an RNAi-resistant USP37, but not the catalytically impaired USP37(C350A) mutant. Further, USP37 associates with WAPL, a negative regulator of cohesion necessary for cohesin release in prophase, in a manner dependent on USP37's second and third ubiquitin-interacting motifs. Depletion of USP37 reduces the stability of chromatin-associated WAPL and increases the fraction of WAPL that is more heavily ubiquitylated in mitosis. Consistently, overexpression of USP37(C350A) results in increased modification of WAPL, and addition of purified USP37(WT), but not USP37(C350A), to WAPL immunoprecipitates results in a reduction of ubiquitylated products. Taken together, our results ascribe a novel function for USP37 in mitotic progression and further suggest that USP37 positively regulates the stability of chromatin-associated WAPL to facilitate sister chromatid resolution.