Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3.
Biomolecules
; 12(1)2022 01 01.
Article
em En
| MEDLINE
| ID: mdl-35053210
ABSTRACT
There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Substituição de Aminoácidos
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Mutação de Sentido Incorreto
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Ubiquitina
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Ubiquitina Tiolesterase
Limite:
Humans
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article