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

Abstract

Post-translational modifications with ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are regulated by isopeptidases termed deubiquitinases (DUBs) and Ubl proteases. Here, we describe a mild chemical method for the preparation of fluorescence polarization substrates for these enzymes that is based on the activation of C-terminal Ub/Ubl hydrazides to acyl azides and their subsequent functionalization to isopeptides. The procedure is complemented by native purification routes and thus circumvents the previous need for desulfurization and refolding. Its broad applicability was demonstrated by the generation of fully cleavable substrates for Ub, SUMO1, SUMO2, NEDD8, ISG15, and Fubi. We employed these reagents for the investigation of substrate specificities of human UCHL3, USPL1, USP2, USP7, USP16, USP18, and USP36. Pronounced selectivity of USPL1 for SUMO2/3 over SUMO1 was observed, which we rationalize with crystal structures and biochemical assays, revealing a SUMO paralogue specificity mechanism distinct from SENP family deSUMOylases. Moreover, we investigated the recently identified Fubi proteases USP16 and USP36 and found both to act as bona fide deFubiylases, harboring catalytic activity against isopeptide-linked Fubi. Surprisingly, we also noticed the activity of both enzymes toward ISG15, previously not identified in chemoproteomics, which makes USP16 and USP36 the first human DUBs with specific isopeptidase activity toward three distinct modifiers. The methods described here for the preparation of isopeptide-linked, fully folded substrates will aid in the characterization of further DUBs/Ubl proteases. More broadly, our findings highlight possible limitations associated with fluorogenic substrates and Ubl activity-based probes and stress the importance of isopeptide-containing reagents for validating isopeptidase activities and quantifying substrate specificities.

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

“…Modification of substrate proteins at target lysine residues with ISG15 is through an enzymatic cascade via an E1 (UBA7/UbE1L), E2 (UBE2L6/UBCH8), and one of three E3 ligases (HERC5, HHARI, EFP/TRIM25). USP18 [ 275 , 276 ], along with USP5, USP14, USP21 and recently USP16 and USP36, have been identified to have deISGylase activity to reverse the modification [ 277 ]. Critically, expression of ISG15 and the ISGylation machinery is stimulated by type I interferons, cytoplasmic nucleic acid detection pathways, and other cellular stresses, with ISG15 predominantly known for its role in the classical innate immune response during viral infections to limit viral replication [ 30 , 278 , 279 ].…”

“…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.…”

“…Progress in mass spectrometry techniques and proteomics analysis, alongside diglycine-enrichment and other tools, has allowed detection of the Ub/UBL linkages relevant to DNA damage signalling. Coupling this to semi-synthetic and designer approaches for producing Ub/UBL substrates, such as for UBLs [ 277 ] and mixed/branched chains [ 309 , 310 ], has helped to clarify the activity and specificity of DUBs and ULPs. Further enhancement in structure prediction using AlphaFold [ 311–314 ] and the rapid progress in structural biology using cryo-EM alongside complementary techniques, has provided hitherto unmatched detail regarding the regulation and activity of DUBs/ULPs.…”

In the moonlight: non-catalytic functions of ubiquitin and ubiquitin-like proteases

“…Although the classification into the seven families mentioned above is the most commonly used, it has to be pointed out that names of DUBs and UBL proteases might be misleading in several cases. Examples include: (1) USP18, which despite its name ( Ubiquitin-specific protease 18 ) does not recognize nor cleave ubiquitin but specifically deconjugates solely ISG15; (2) USP2, USP5, USP14 and USP21, which not exclusively cleave ubiquitinated substrates but also ISGylated proteins; (3) USP16 and USP36, which display cross-reactivity between ubiquitin, FUBI and ISG15 ( Zhao et al, 2023 ) and (4) SENP8, which despite its name does not cleave SUMO at all but exhibits specificity for NEDD8 instead ( Kunz et al, 2018 ).…”

Triboelectric Spectroscopy for In Situ Chemical Analysis of Liquids

“…Chemical analysis is central to all fields of science, technology, engineering, and health. − Strategies for the analysis of ions and organics include optical, electrochemical, and chromatographic methods . Optical methods often lack selectivity and generally require large and expensive instrumentation, and mainstream chromatographic instruments remain far from portable and inexpensive .…”