Proteome-wide ubiquitinome profiling reveals substrate-specific dynamics within the USP7 network
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
ABSTRACT USP7 is a pleiotropic deubiquitylating enzyme that is involved in tumor suppression, (neuro)development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we report data-independent acquisition and label free quantitation mass spectrometry (DIA-LFQ-MS) to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. First, we identified proteins associated with endogenous USP7 by immunopurification followed by DIA-LFQ-MS. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence protein targets. Domain mapping analysis revealed that, in addition to the TRAF domain, the ubiquitin-like domains of USP7 play a key role in substrate selection. Using specific enrichment of tryptic K-ε-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have different effects on the stability of distinct substrates, and suggests that USP7’s activity profile is substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach reveals that the effects of USP7-mediated deubiquitylation on its targets are remarkably variable and substrate-specific. Finally, based on these detailed molecular insights we show how USP7 connects various neurodevelopmental syndromes and tumor suppression pathways.
Authors 8
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Affiliation as printed
Department of Developmental Biology, Erasmus University Medical Center, Rotterdam, The Netherlands
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Affiliation as printed
Department of Developmental Biology, Erasmus University Medical Center, Rotterdam, The Netherlands
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Affiliation as printed
Department of Developmental Biology, Erasmus University Medical Center, Rotterdam, The Netherlands
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Affiliation as printed
Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands
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Affiliation as printed
Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands
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Affiliation as printed
Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands
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Jeroen Demmers Aachen Leiden Academic Centre for Drug Research Faculty of Science, Leiden University
Leiden University · Erasmus MC
Affiliation as printed
Leiden Academic Center for Drug Research, Faculty of Science, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands
Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands
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Affiliation as printed
Department of Developmental Biology, Erasmus University Medical Center, Rotterdam, The Netherlands
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