Proteomics-Based Identification of DUB Substrates Using Selective Inhibitors

Abstract

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.

Keywords: DUBs; chemical probe; deubiquitinating enzymes; drug discovery; proteomics.

Figure 1.. Differential Proteomics Analysis of MM.1S Cells Treated with Selective USP7 Inhibitors

(A) In vitro biochemical selectivity of XL series USP7 inhibitors. Compounds XL188, XL203C, and XL177A were added to in vitro deubiquitination assays at concentrations of 10, 10, and 1 μM, respectively. (B) Scatterplot representation of protein abundance changes after USP7 inhibitor treatment. Orange points represent proteins that pass the threshold of >1.5-fold change and p < 0.0001. p values and log₂FC values were calculated using a moderated t test as implemented in limma. See Data S1 in the Supplemental Information for matching tables. (C) Rank-order representation of protein abundance changes after USP7 inhibition. Proteins were ranked in ascending order using p value-adjusted log₂ fold change as a score (Pi score = −log₁₀(p value) × log₂FC). Thresholds same as in (B).

Figure 2.. Time Course of Protein Abundance Changes Following USP7 Inhibition

(A) Scatterplot representation of protein abundance changes over time with XL177A (see Figure S2 for XL188). Thresholds and statistical test are the same as in Figure 1. (B) Heatmap of log₂FC protein abundance values for differentially quantified proteins following USP7 inhibition by XL177A or XL188 after either 2, 6, 12, or 24 h treatment. Proteins indicated in bold were differentially quantified after 2 h XL177A treatment. See Data S1 in the Supplemental Information for matching tables.

Figure 3.. Evaluation of High-Confidence USP7 Candidate Substrates

(A) Heatmap of Pi scores for top differentially quantified proteins and known USP7 targets (see Table S1) following USP7 inhibition. (B) Heatmap comparison of USP7 inhibition against USP7 IP-MS data from the DUB Interactome database (see Figure S3A for Pearson correlation, Figure S3B for BioPlex database). (C) STRING network analysis of proteins destabilized by XL177A and XL188 treatment (D) Annotation of USP7 targets with enriched Gene Ontology molecular function terms. Gene enrichment was calculated using Fisher’s exact test as implemented in Enrichr.

Figure 4.. Differential Proteomics Analysis of Additional DUB Inhibitors

(A) Comparison of singlicate (3× DMSO versus 1× Drug) and triplicate (3× DMSO versus 3× Drug) analysis of protein abundance changes after thalidomide treatment. (B) Rank-order representation of differentially quantified proteins (as in Figure 1C) after singlicate cell treatments with various dual-selective DUB inhibitors. Thresholds same as Figures 1 and 2. See Data S1 and Data S2 in the Supplemental Information for matching tables and scatterplots. (C) Heatmap comparison of proteins disrupted by DUB inhibition in two independent treatment doses or experiments.