Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin- and Ubl-proteases collectively called DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity towards ubiquitin but specifically deconjugates the interferon induced Ubl ISG15. To identify molecular determinants for this specificity, we solved the crystal structures of mouse USP18 and of mouse USP18 in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15 providing evidence that ISG15 specificity of USP18 is mediated by a small interaction interface. Our results may provide the structural basis for the development of new drugs modulating ISGylation.

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades.

Deubiquitinating enzymes (DUBs) regulate ubiquitin signaling by trimming ubiquitin chains or removing ubiquitin from modified substrates. Similar activities exist for ubiquitin-related modifiers, although the enzymes involved are usually not related. Here, we report human ZUFSP (also known as ZUP1 and C6orf113) and fission yeast Mug105 as founding members of a DUB family different from the six known DUB classes. The crystal structure of human ZUFSP in covalent complex with propargylated ubiquitin shows that the DUB family shares a fold with UFM1- and Atg8-specific proteases, but uses a different active site more similar to canonical DUB enzymes. ZUFSP family members differ widely in linkage specificity through differential use of modular ubiquitin-binding domains (UBDs). While the minimalistic Mug105 prefers K48 chains, ZUFSP uses multiple UBDs for its K63-specific endo-DUB activity. K63 specificity, localization, and protein interaction network suggest a role for ZUFSP in DNA damage response.

Drugs that increase 26S proteasome activity have potential therapeutic applications in the treatment of neurodegenerative diseases. A chemical genetics screen of over 2,750 compounds using a proteasome activity probe as a readout in a high-throughput live-cell fluorescence-activated cell sorting-based assay revealed more than ten compounds that increase proteasome activity, with the p38 MAPK inhibitor PD169316 being one of the most potent ones. Genetic and chemical inhibition of either p38 MAPK, its upstream regulators, ASK1 and MKK6, and downstream target, MK2, enhance proteasome activity. Chemical activation of the 26S proteasome increases PROTAC-mediated and ubiquitin-dependent protein degradation and decreases the levels of both overexpressed and endogenous α-synuclein, without affecting the overall protein turnover. In addition, survival of cells overexpressing toxic α-synuclein assemblies is increased in the presence of p38 MAPK inhibitors. These findings highlight the potential of activation of 26S proteasome activity and that this can be achieved through multiple mechanisms by distinct molecules.