Investigating Short Linear Motif (SLiM)-Deubiquitinase interactions in modulating ubiquitin signalling

Deubiquitylating enzymes (DUBs) represent an essential regulatory layer in maintaining cellular homeostasis and are often dysregulated in diseases. By countering ubiquitylation, one of the most abundant post-translational modifications, DUBs play diverse roles across various cellular pathways. The mechanisms underlying substrate recognition and specificity by DUBs remain unclear. While some DUBs exhibit selectivity for specific ubiquitin linkage types, others demonstrate substrate-specific recognition. The central question of my PhD research was whether Short Linear Motifs (SLiMs) mediate substrate targeting in DUBs. SLiMs are enriched within the intrinsically disordered regions of eukaryotic proteomes. They function as binding interfaces for protein complex assembly (scaffolds), guide proteins to subcellular compartments, mediate the docking of enzymes to their substrates, and serve as degradation sites (degrons) targeted by E3 ligases. SLiMs have been identified for enzyme families such as kinases, phosphatases, and E3 ligases, but not for deubiquitinases (DUBs). Hence, I aimed to screen recombinant DUBs against intrinsically disordered regions of eukaryotic and viral proteomes to identify motifs recognised by DUBs. To ensure a comprehensive screening approach, I first investigated the existence of any undiscovered DUBs by combining AlphaFold and the DALI server as discovery tools. This analysis identified 16 proteins with folds similar to DUBs that also contain a catalytic triad. Despite their structural similarity, these proteins did not exhibit activity toward ubiquitin. Subsequently, I screened 71 DUBs against the HD2 phage display library, which comprises 938,427 peptides derived from disordered regions of 16,969 proteins, representing approximately one-third of the proteome with overlapping peptides. Using this large-scale approach and deep mutational analysis, I identified consensus motifs recognised by ZUP1, USP7, USP20, USP33, OTUD7B, AMSH-LP, CYLD, and UCHL5.