Comprehensive Landscape of Active Deubiquitinating Enzymes Profiled by Advanced Chemoproteomics

Abstract

Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.

Keywords: chemical biology; deubiquitylating enzymes; isoforms; mass spectrometry; proteomics; ubiquitin specific proteases.

Figure 1

DUB transcriptome and proteome in MCF7 breast cancer cells. (A) Transcriptome analysis (single experiment data) listing all quantified mRNAs as reads per Kilobase per million (RPKM) values in descending values. mRNAs encoding DUBs are indicated according to their families: 56 USPs (red), 5 UCHs (dark green), 16 OTUs (blue), 11 JAMMs (purple), 4 MINDYs (yellow), 4 JOS (light green), and 1 ZUP (black). (B) Proteome analysis (single experiment data) listing all quantified proteins as intensity-based absolute quantitation (iBAQ) abundances in descending values. DUBs are indicated and colored based on sub-families as stated above. (C) Scatter Plot showing mRNA (transcriptome, X-axis) and protein (proteome, Y-axis) levels of DUBs (indicated in colors according to sub-families).

Figure 2

Ub-PA probe chemistry extends DUBome activity-based profiling. (A) Titration of HA-UbC2Br (left panel) and HA-UbPA probe (right panel) in MCF7 breast cancer cell extracts, followed by SDS-PAGE separation and analysis by anti-HA immunoblotting. Bands correspond to either DUB-probe or E3 ligase-probe adducts as indicated [based on (Altun et al., 2011) and this study]. (B) Left panel: Chemoproteomics workflow for profiling the active DUBome. HA-UbC2Br or HA-UbPA probe is incubated with MCF7 breast cancer cell extracts, followed by anti-HA immunoprecipitation, elution, in-solution trypsin digestion, and label-free quantitative analysis (LFQ) by LC-MS/MS. Right panel: Comparison of HA-UbC2Br and HA-UbPA immunoprecipitated DUBs analyzed by SDS-PAGE and anti-HA, anti-USP7 (positive control), and anti-GAPDH (loading control) immunoblotting.

Figure 3

Full DUBome coverage by advanced chemoproteomics. Scatter Plot showing enrichment of DUBs upon labeling and isolation of HA-UbPA activity-based probe pulldown and quantitative analysis by mass spectrometry (concatenation of 60 fractions into 10). X-axis—no probe; Y-axis—with probe. The experimental workflow is shown on the top. The graph inclusion shows the number of DUBs captured by the HA-UbPA probe with (single experiment data) and without (technical triplicates) high-pH prefractionation post-HA-IP and digestion.

Figure 4

Mapping direct Ub–probe DUB adducts by mass spectrometry. LC-MS/MS analysis of HA-UbPA-labeled DUBs isolated from MCF7 breast cancer cell extracts. Peptide mapping using PEAKS analysis reveals direct cysteine–probe adducts (light blue boxes—P) for the DUBs UCHL3, OTUB1, OTUD3, OTUD4, and OTUD6B. The corresponding MS/MS fragmentation spectra for assigning the Cys–UbPA–probe adducts are listed in Figures S3A–S3E.

Figure 5

Active vs. non-active DUBs. Volcano plots showing a discrimination between cysteine-reactive and non-reactive DUBs by active-site labeling with HA-UbPA in the presence and absence of N-ethylmaleimide (NEM, Left panel) and PR-619 (Right panel) (data from two biological replicates run in technical duplicates). Displaced reactive DUBs are located in the upper left compartment ([probe alone]—[probe + NEM or PR619]), whereas non-reacting DUBs are left in the lower center area.

Figure 6

MCF7 DUB activitome vs. transcriptome and proteome. Scatter Plots showing the correlative traits of the transcriptome (A) and the proteome (B) with the DUB activitome (X-axis in both panels). DUBs are indicated in colors respective to their enzyme sub-families.

Figure 7

Expanded panel of active DUBs across the different enzyme subfamilies. ML (maximum likelihood) phylogenetic tree of the human DUB family. One hundred and two DUBs of the ubiquitin specific protease (USP), ubiquitin C-terminal hydrolases (UCH), ovarian tumor domain (OTU), JAMM, MINDY, JOS, and ZUP1 families are shown. In bold are the DUBs detected in the cellular proteome by active-site labeling, indicating cellular activity. *indicates inactive DUBs, in part because of mutated catalytic site cysteines and **indicates selective induction by type I interferon (IFN). The gene name nomenclature was used for consistency, but many DUBs have alternative names: ZA20D1 (OTU7B), YOD1 (OTU1/DUBA8), TNFAIP3 (OTUD7C/A20), AMSH (STAMBP1), AMSH-like (STAMBPL1), TL132 (USP32P2), TL132-like (USP32P1), LOC339799 (EIF3FP3), ZRANB1 (TRABID), PSMD14 (RPN11/POH1), USP17L2 (DUB3), OTUD6B (DUBA5, CGI-77), and PAN2 (USP52). In bold are those DUBs detected in the proteome, and the (*) indicates DUBs reactive to UbPA probe.