A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

Abstract

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.

Mechanism and disease association of E2-conjugating enzymes: lessons from UBE2T and UBE2L3

“…For example, UBE2N is an emerging key player in various cancers, and the recently developed small compound inhibitor NSC697923 targeting UBE2N inhibits proliferation and survival of neuroblastoma and DLBCL cells. With the advent of enzymatic tools for trapping various states of E2 intermediates, there are now more opportunities to address the unique properties of E2s in different biological contexts and, importantly, to dissect disease pathways [189,190]. Certainly, this will pave the way to exploit E2s in therapeutic contexts, in particular by developing new strategies to target individual E2s or specific interactions of E2–E3 pairs.…”

Generation of the UFM1 Toolkit for Profiling UFM1‐Specific Proteases and Ligases

“…Notwithstanding the biochemical and structural studies of UFM1‐conjugating and deconjugating enzymes that have been undertaken,17, 18, 19, 20, 21 their biological function remains enigmatic primarily owing to the lack of activity‐based reagents. By contrast, diverse reagents and ABPs have been developed for both Ub‐conjugating and deconjugating enzymes22, 23, 24, 25, 26, 27 and have been expanded to Ubls such as SUMO28, 29, 30 and Nedd8 24, 26. This advancement of assay and activity‐based reagents has greatly propelled discoveries in the ubiquitin field, yet such a diversifiable synthetic platform for UFM1 needs to be developed 31.…”

“…For this purpose, we synthesized UFM1‐Dha to target the conjugating class of enzymes26 and UFM1‐PA to target the proteases 24. To achieve this, we synthesized a C‐terminal fragment lacking the last amino acid (A45C‐82).…”

“…Subsequently, we addressed the efficacy of UFM1‐PA by monitoring UFM1 protease activity in a cellular environment by electroporation of Rho‐UFM1‐PA ( 10 ) into HeLa cells ectopically expressing Flag‐Ufsp1 or its catalytically inactive C53A mutant as previously described 26. In‐gel fluorescence analysis followed by immunoblotting revealed labeling of endogenous Ufsp2 in cells with Rho‐UFM1‐PA, while in Ufsp2‐depleted HeLa cells reactivity was abolished (see Figure 5 A and Figures S9 and S10).…”

Selenocysteine as a Latent Bioorthogonal Electrophilic Probe for Deubiquitylating Enzymes

“…5–7 However, the challenges associated with their chemoselective installation limits their application to proteins that lack cysteine. For example, the Michael acceptor dehydroalanine (Dha) was recently reported as a probe for DUB activity.…”