Novel Diubiquitin Probes Expand the Chemical Toolkit to Study DUBs.

Expanding the chemical toolkit to study DUBs with novel diubiquitin probes

Activity-based diubiquitin probes for elucidating the linkage specificity of deubiquitinating enzymes.

We report a new class of deubiquitinating enzyme (DUB) probes that resemble the native diubiquitin with a same linkage size and contain a Michael addition acceptor for trapping the DUB active-site cysteine. Both K63- and K48-linked diubiquitin probes were generated using a facile chemical ligation method. The diUb probes were demonstrated to label DUBs from different families and revealed intri...

Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets.

Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer speci...

Development of Diubiquitin‐Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes

Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the a...

A Native Chemical Ligation Handle that Enables the Synthesis of Advanced Activity-Based Probes: Diubiquitin as a Case Study

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity-based probes. We describe the use of this handle in the generation of linkage-specific activity-based deubiquitylating enzyme probes that contain substrate context and...