Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

Abstract

Ubiquitin and ubiquitin-like proteins typically use distinct machineries to facilitate diverse functions. The immunosuppressive ubiquitin-like protein Fubi is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30). Its proteolytic maturation by the nucleolar deubiquitinase USP36 is strictly required for translationally competent ribosomes. What endows USP36 with this activity, how Fubi is recognized and whether other Fubi proteases exist are unclear. Here, we report a chemical tool kit that facilitated the discovery of dual ubiquitin/Fubi cleavage activity in USP16 in addition to USP36 by chemoproteomics. Crystal structures of USP36 complexed with Fubi and ubiquitin uncover its substrate recognition mechanism and explain how other deubiquitinases are restricted from Fubi. Furthermore, we introduce Fubi C-terminal hydrolase measurements and reveal a synergistic role of USP16 in Fubi-S30 maturation. Our data highlight how ubiquitin/Fubi specificity is achieved in a subset of human deubiquitinases and open the door to a systematic investigation of the Fubi system.

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases

“…We therefore turned to the Ubl Fubi which like ubiquitin is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30) and cleaved by the nucleolar DUB USP36 to release free Fubi and S30 . In addition to USP36, the mainly cytosolic DUB USP16 has Fubi protease activity, which may give rise to a two-tier system of Fubi-S30 maturation . In immune cells, Fubi conveys immunosuppressive signaling, including suppression of the maternal immune system upon embryo implantation, and isopeptide-linked conjugates of Fubi have been observed for selected proteins (termed Fubiylation in analogy to Ubiquitylation). , However, which enzymes can act as deFubiylases with the ability to specifically antagonize these isopeptide-linked Fubi conjugates is not known.…”

“…To address these shortcomings, we developed individual synthesis and purification protocols for the generation of fully folded isopeptide-linked fluorescence polarization substrates based on Ubl C-terminal acyl azides. We confirm the complete conversion of a suite of Ub/Ubl-KG-TAMRA reagents in enzymatic assays and apply it to the quantification of Ub/Ubl cross-reactivity of various DUBs including the recently identified Fubi proteases USP16 and USP36, , which we show to be the first trispecific Ubl isopeptidase with previously overlooked deISGylation activity. Moreover, we focused on recently structurally characterized USPL1 (Figure D), which is the only deSUMOylase with a USP fold but for which its mechanism for SUMO paralogue specificity had remained unclear.…”

“…There are approximately 100 DUBs known in humans which can be grouped into 7 different classes of which the ubiquitin-specific proteases (USPs) are the largest and most heterogeneous family (Figure C) . While the bulk of USP DUBs are considered to be indeed ubiquitin-specific yet rather promiscuous with regards to the ubiquitinated substrates, some members notably feature preferences for distinct ubiquitin chains (CYLD and USP30), display Ub/Ubl cross-reactivity (USP16 and USP36 for Ub and the Ubl Fubi; , USP2, USP5, USP14 and USP21 for Ubiquitin and ISG15 − ), or are specific for a Ubl without ubiquitin activity (USP18 for ISG15, USPL1 for SUMO, Figure D). Ub/Ubl cross-reactivity has also been observed in other DUB families (e.g., UCHL3 with activity for Ub and NEDD8) and is particularly prevalent in viral and bacterial effector DUBs. − Moreover, additional examples exist where a member of a particular Ubl protease fold has evolutionarily been co-opted to provide cleavage activity for a distinct Ubl.…”

Surprising variety in the USP deubiquitinase catalytic mechanism

“…As serine is highly efficient at forming H-bonds, it could be that this serine is the critical residue of choice in USP16 and USP45 as well. Recently, cellular USP45 was shown to display elevated reactivity towards protein probes, not in line with its in vitro catalytic activity, which stresses that serine as the third catalytic residue does not coincide with lower nucleophilicity (O'Dea et al, 2023).…”

DoUBLing up: ubiquitin and ubiquitin-like proteases in genome stability

“…USP18 is predominantly the deISGylase thought to act in cells particularly as its expression aligns with the interferon response and ISG15 . Other USP-family members such as USP5, USP14, USP16, USP21, and USP36 [ 277 , 287 ], have all shown deISGylase activity in vitro on top of their primary protease activity against other UBLs and/or Ub. Whether these moonlighting activities are relevant to ISG15 biology remains to be seen, but the development of specific tools to probe activity in response to DNA damage will likely aid future work.…”