Strategies to Target Specific Components of the Ubiquitin Conjugation/Deconjugation Machinery

Abstract

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery.

Exploring the Versatility of the Covalent Thiol–Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease

“…Another possibility would be to introduce primed site recognition peptide fragments on the terminal alkyne position to improve selectivity and/or affinity. 10 , 65 , 66 …”

“…Another possibility would be to introduce primed site recognition peptide fragments on the terminal alkyne position to improve selectivity and/or affinity. 10,65,66 The reaction mechanism (Scheme 1B or 1D) has extensive consequences for the scope of the in situ alkyne−thiol addition. Enzyme-templated stabilization of a carbanion intermediate (Scheme 1D) would restrict the applicability in drug design to targeting catalytic cysteine residues with unactivated alkynes, but it also mitigates the risk of covalent adduct formation with nontargeted thiols.…”

“…There are currently six known classes of human cysteine DUBs: USP, OTU, UCH, MJD, MINDY, and ZUFSP. 1 , 10 Their proteolytic activity can be monitored with activity-based probes (ABPs), which covalently trap active enzymes by formation of a covalent bond between an electrophilic warhead on the ABP and the nucleophilic cysteine residue in the targeted enzyme. 11 − 13 Cysteine DUB ABPs have been utilized to monitor DUB activity during infection, in disease and/or upon inhibitor treatment, 14 − 17 to identify new DUB (classes) and catalytic cysteine residues in newly discovered DUBs, 18 − 21 and to visualize Ub binding in crystal structures of covalent adducts.…”

“…Deubiquitinating enzymes (DUBs) reverse this process by cleavage of the native isopeptide bond between the Ub C-terminus and the target protein Lys (lysine) residue or between the distal and proximal Ub in poly-Ub chains. , Cysteine DUBs are classified by their catalytic domain, which contains a catalytic cysteine residue essential for their proteolytic function. There are currently six known classes of human cysteine DUBs: USP, OTU, UCH, MJD, MINDY, and ZUFSP. , Their proteolytic activity can be monitored with activity-based probes (ABPs), which covalently trap active enzymes by formation of a covalent bond between an electrophilic warhead on the ABP and the nucleophilic cysteine residue in the targeted enzyme. − Cysteine DUB ABPs have been utilized to monitor DUB activity during infection, in disease and/or upon inhibitor treatment, − to identify new DUB (classes) and catalytic cysteine residues in newly discovered DUBs, − and to visualize Ub binding in crystal structures of covalent adducts. , …”

Flipping the Switch: Innovations in Inducible Probes for Protein Profiling

“…While activity-based probes have been used to probe a diverse range of active enzymes, ,− applications of these tool compounds can have limitations. Highly electrophilic warheads lead to nonspecific protein labeling due to off-target reactivity .…”