Dataset Information

USP7: combing tools towards selectivity

ABSTRACT: Active-site probes based on the ubiquitin scaffold have been successfully applied as tools to determine the levels of active deubiquitinating enzymes (DUBs). Here, we show the development of a Ub-based reagent selective for the DUB USP7. This concept can be applied for the generation of other new selective reagents.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Román González-Prieto

LAB HEAD: Alfred C.O. Vertegaal

PROVIDER: PXD012535 | Pride | 2019-03-13

REPOSITORIES: Pride

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USP7: combining tools towards selectivity.

Gjonaj Lorina L Sapmaz Aysegul A González-Prieto Román R Vertegaal Alfred C O ACO Flierman Dennis D Ovaa Huib H

Chemical communications (Cambridge, England) 20190401 35

Active-site directed probes based on the ubiquitin scaffold have been successfully applied as tools to determine the levels of active deubiquitinating enzymes (DUBs). Here, we show the development of a Ub-based reagent selective for the DUB USP7. This concept can be applied for the generation of other new selective reagents. ...[more]

PMID: 30972393

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Project description:Genotoxic agents can cause replication fork stalling in dividing cells due to DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. The methodology is generic and is widely applicable in the ubiquitin field. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 hours of Hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and 2 proteins were down-regulated for SUMOylation, whereas after 24 hours, 35 proteins were up-regulated for SUMOylation and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1,000 SUMO-2 acceptor lysines in target proteins. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1 and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the aim of dealing with the induced DNA damage

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