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.

Keywords: activity-based protein profiling; deubiquitinating enzymes; probe; protein modification; ubiquitin.

Figure 1

Retrosynthesis of selected probes. (A) A methodology reported by Li et al. utilizes an α-bromo-vinylketal to link Ub₇₅ thioester 6 and the mutated Ub monomer 4. Deprotection of the ketal of 2 unmasks a Michael acceptor within the linker of the probe 1. (B) Haj-Yahya et al. synthesized a diubiquitin probe based on DHA as the electrophilic warhead. NCL is used to link Ub monomers 6 and 10, positioning a cysteine residue at position 76 of the distal Ub which is then converted to DHA using the dibromide reagent 7. (C) Pao et al. expanded on the TDAE methodology to incorporate a Ub monomer and E2 enzyme in a single probe. Alkyne functionalized TDAE 13 is coupled to azido functionalized Ub monomer 14 using copper catalyzed cycloaddition. A subsequent reaction with an E2 enzyme eliminates the tosyl component of the TDAE 12, affording the final probe 11 containing a Michael acceptor. (D) Flieman et al. use copper catalyzed cycloaddition to conjugate two modified Ub monomers 19 and 20. Propargyl amine 17 was reacted with the C-terminus of the proximal monomer to yield probe 16.

Figure 2

A selection of ubiquitin-based probes that specifically target subsets of DUBs (blue), E1s (black) and E3s (green) compared to the natural substrates of their targets (framed).