Advancing the Exploration of the Ubiquitin-like Protein FUBI with Synthetic Chemical Tools.

The Ubiquitin-like protein FUBI is encoded in humans by the FAU gene, whose down-regulation in prostate, ovarian and breast cancer is significantly associated with poor prognosis. Despite its implications in disease progression, the regulatory mechanisms orchestrated by FUBI remain elusive. To address this knowledge gap, a linear synthetic platform is developed to generate FUBI chemical tools, enabling the site-specific incorporation of unnatural building blocks and the introduction of fluorophores, tags, and reactive warheads. Using this platform, activity-based probes are created for FUBI conjugation and deconjugation enzymes, validating them in cell lysate-based assays and proteomics. Additionally, a triazole-linked Di-FUBI is synthesized to investigate FUBI chain modulators. Among the proteomics hits, IMPDH1 and the deubiquitinase UCHL3 are identified as novel Di-FUBI specific interactors. Further characterization revealed that Di-FUBI inhibits UCHL3 cleavage activity in a concentration-dependent manner, suggesting a novel regulatory interplay between UCHL3 and FUBI. Collectively, these tools demonstrate the versatility of the synthetic FUBI platform, advancing the characterization of FUBI-related enzymes in the ongoing efforts to decipher the complex code of ubiquitin-like signaling.