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bioRxiv · 10.64898/2025.12.29.696849

Proteome-wide ubiquitinome profiling reveals substrate-specific dynamics within the USP7 network

Wolf van der Meer, J.·van der Knaap, J.·Sijm, A.·Bezstarosti, K.·Dekkers, D. H. W.·Doff, W.·Demmers, J. A.·Verrijzer, P.
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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-{varepsilon}-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 USP7s 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.

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Wolf van der Meer, J., van der Knaap, J., Sijm, A., Bezstarosti, K., Dekkers, D. H. W., Doff, W., Demmers, J. A., Verrijzer, P.. 2025-12-29. Proteome-wide ubiquitinome profiling reveals substrate-specific dynamics within the USP7 network. https://doi.org/10.64898/2025.12.29.696849

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