Activity‐Based Probes for HECT E3 Ubiquitin Ligases

Byrne, Robert T.; Mund, Thomas; Licchesi, Julien

doi:10.1002/cbic.201700006

Cited by 14 publications

(20 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“… 7 , 18 We then turned to the biochemical Ub-PA (ubiquitin-propargylamide) assay. In this assay, the catalytic domain of USP14 (USP14 CAT ) was preincubated with IU1, followed by addition of the covalent inhibitor Ub-PA. 19 – 22 The covalent attachment of Ub-PA (8 kD) to USP14 CAT (45 kD) produced the novel USP14/Ub-PA complex with an increased molecular weight (53 kD). We observed that at a 5 mM concentration IU1 significantly inhibited the formation of the USP14/Ub-PA complex (Fig.…”

Section: Resultsmentioning

confidence: 99%

Small molecule inhibitors reveal allosteric regulation of USP14 via steric blockade

et al. 2018

View full text Add to dashboard Cite

The ubiquitin system is important for drug discovery, and the discovery of selective small-molecule inhibitors of deubiquitinating enzymes (DUBs) remains an active yet extremely challenging task. With a few exceptions, previously developed inhibitors have been found to bind the evolutionarily conserved catalytic centers of DUBs, resulting in poor selectivity. The small molecule IU1 was the first-ever specific inhibitor identified and exhibited surprisingly excellent selectivity for USP14 over other DUBs. However, the molecular mechanism for this selectivity was elusive. Herein, we report the high-resolution co-crystal structures of the catalytic domain of USP14 bound to IU1 and three IU1 derivatives. All the structures of these complexes indicate that IU1 and its analogs bind to a previously unknown steric binding site in USP14, thus blocking the access of the C-terminus of ubiquitin to the active site of USP14 and abrogating USP14 activity. Importantly, this steric site in USP14 is very unique, as suggested by structural alignments of USP14 with several known DUB X-ray structures. These results, in conjunction with biochemical characterization, indicate a coherent steric blockade mechanism for USP14 inhibition by compounds of the IU series. In light of the recent report of steric blockade of USP7 by FT671, this work suggests a potential generally applicable allosteric mechanism for the regulation of DUBs via steric blockade, as showcased by our discovery of IU1-248 which is 10-fold more potent than IU1.

show abstract

Section: Resultsmentioning

confidence: 99%

Small molecule inhibitors reveal allosteric regulation of USP14 via steric blockade

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Virdee et al demonstrated an application in profiling primary fibroblasts from Parkinson's disease patients. Licchesi and co-workers (Byrne et al, 2017) later showed these probes react with NEDD4, UBE3C and HECTD1.…”

Section: Ubiquitin-protein Probes For E3 Ligasesmentioning

confidence: 93%

“…Monoubiquitin probes have been demonstrated to label ubiquitin conjugation machinery (Kamadurai et al, 2009;Love et al, 2009;Kim et al, 2011;Ekkebus et al, 2013;Maspero et al, 2013;Byrne et al, 2017) however they lack specificity. Tan and co-workers (Lu et al, 2010) developed E1 targeting probes by mimicking the adenylate intermediate formed in the E1 active site.…”

Section: Ubiquitin-adenine Probesmentioning

confidence: 99%

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

Taylor

McGouran

2020

Front. Chem.

View full text Add to dashboard Cite

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.

show abstract

“…Thus, the E3 was named a RING-Cys-relay E3, and it is the first E3 shown to have a preference for Thr ubiquitination. Similar design of E2-UB conjugates with an acrylamide linker as a Cys trap has been used to probe HECT E3 activity in cell lysates (Byrne et al, 2017). E2-UB probes demonstrate the power of protein engineering to generate customized tools to discover new cellular protein ubiquitination activities.…”

Section: B Activity-based Probes For Ubiquitin-transferringmentioning

confidence: 99%