Proteome-wide Ubiquitinome Profiling Reveals Substrate-specific Dynamics Within the USP7 Network

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• USP7 is a deubiquitylating enzyme crucial for tumor suppression, neurodevelopment, chromatin regulation, and DNA damage response.
• The precise mechanisms by which USP7 regulates these diverse cellular pathways remain incompletely understood.

Purpose of the Study:

• To comprehensively profile the proteome-wide effects of USP7 on substrate deubiquitylation and protein abundance.
• To elucidate the role of USP7 domains in substrate selection and understand the substrate-specific nature of its deubiquitylation activity.

Main Methods:

• Utilized data-independent acquisition and label-free quantitation mass spectrometry (DIA-LFQ-MS) to profile the proteome.
• Employed immunopurification to identify endogenous USP7-associated proteins.
• Mapped proteome-wide changes in ubiquitination dynamics using K-ε-GG peptide enrichment after USP7 inhibition.

Main Results:

• Identified a consensus set of high-confidence USP7 protein targets by integrating new and existing interactome data.
• Revealed that USP7's ubiquitin-like domains, in addition to its TRAF domain, are critical for substrate recognition.
• Demonstrated that USP7-mediated deubiquitylation has variable and substrate-specific effects on target protein stability.
• Established that USP7's activity profile is substrate-dependent, not an intrinsic enzymatic property.

Conclusions:

• USP7's deubiquitylation activity is highly variable and substrate-specific, influencing diverse pathways including neurodevelopment and tumor suppression.
• Provides a proteome-wide map of USP7 target sites and insights into its regulatory mechanisms.
• Highlights the connection between USP7, neurodevelopmental syndromes, and tumor suppression pathways.