Reactive-site-centric chemoproteomics identifies a distinct class of deubiquitinase enzymes

Abstract

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

Fig. 1

Conventional DUB ABPs label DUBs on both catalytic and non-catalytic Cys residues. a Structure of probe-labeled peptide fragment generated by trypsin digestion of Ub-VS-labeled proteins. In these ABPs, an HA tag is attached to residues 1–75 of ubiquitin by a spacer of two aminohexanoyl units. b Detection of probe-labeled DUB peptides in HEK 293T lysate after anti-HA immunoprecipitation and filter-assisted sample preparation (FASP), using HA-tagged Ub-VS, Ub-VME, or Ub-PA probes. Peptides were searched using the probe fragment as a variable modification on Cys. (Modification on other residues could not be detected.) Non-catalytic Cys residues are shown in red. # = Ub-probe fragment; * = carbamidomethyl modification; @ = methionine oxidation. c Reactive-site-centric chemoproteomic strategy to identify probe targets and labeling sites. Lysates are treated with an alkyne-containing ABP. Click chemistry is used to attach a cleavable biotin-azide tag and tagged proteins are enriched on streptavidin beads, using stringent washing to remove non-covalent binders. Proteins are digested on-bead to provide peptides for protein identification. The beads are then subject either to immediate linker cleavage to release probe-labeled peptides or to a second proteolysis before linker cleavage. The yellow triangle represents an electrophilic group, the pale blue rectangle represents a cleavable linker, and the green circle represents biotin

Fig. 2

Design and reactivity of alkyne-containing DUB probes. a Structures of probes used in this work. The vinyl pentynyl sulfone (VPS) and vinyl pentynyl ester (VPE) probes maintain the electrophilic groups of the commonly-used vinyl sulfone (VS) and vinyl methyl ester (VME) probes, and additionally contain an alkyne for attachment to biotin. Ubl, ubiquitin/ubiquitin-like protein. b Peptide-spectrum matches (PSMs) for DUBs detected following anti-HA IP, using either Ub-VS or Ub-VPS probes. Data are representative of two biological replicates. c Structures of linkers used in this work. d Western blottings (or streptavidin-biotin blotting) indicate the effectiveness of labeling with Ub-VPS, tagging with biotin-DADPS-azide, precipitation, and enrichment

Fig. 3

A reactive-site-centric chemoproteomic method to identify probe-labeling sites. a Comparison of the number of DUBs identified by different purification or digestion methods. HEK 293T lysate was treated with Ub-VS (condition 1) or Ub-VPS (conditions 2 and 3), and then subject to anti-HA IP followed by FASP using trypsin (condition 1) or tagged with biotin-DADPS-azide, enriched on streptavidin (SA) beads, and digested on-bead with trypsin (condition 2) or Lys-C and trypsin (condition 3). Shown is the number of DUBs identified with ≥ 3 unique peptides, in each of two or three biological replicates (indicated with Roman numerals). b Volcano plot of pairwise comparison of proteins in on-bead digestion fraction from ABPP using Ub-VPS probe and SUMO2-VPS probe. Statistical significance (− log₁₀ p-value) is plotted against fold enrichment (average log₂) from three biological replicates. A black border indicates proteins with high-confidence probe-labeling sites (i.e., sites identified in more than one experiment). ZUFSP is indicated with an arrow. c Comparison of the number of labeled proteins identified after cleavage of DADPS linker using different combinations of proteases. Shown is the number of proteins for which one or more labeled peptides were detected in any one of six biological replicates. Each replicate corresponds to a nominal protein input of 3 mg. d Venn diagram showing the intersections of probe-labeling sites identified using different combinations of proteases. Diagram produced using Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/)

Fig. 4

Ub-VPS labels UCHL1 on multiple sites. a Sites of UCHL1 labeling identified by ABPP with Ub-VPS using different digestion conditions. b X-ray crystal structure of UCHL1 (green) bound to Ub-VME (purple). PDB ID: 3ifw. Labeled Cys residues are indicated in space-filling representation. c Reactivity of recombinant UCHL1 mutants toward Ub-VS. UCHL1 or the indicated mutants were treated with probe, then analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue

Fig. 5

ZUFSP interacts with ubiquitin and reacts with Ub ABPs. a Domain structure of ZUFSP. Domain prediction from SMART (http://smart.embl-heidelberg.de). Figure produced using Illustrator for Biological Sequences (http://ibs.biocuckoo.org). b A representative MS/MS spectrum showing a peptide-spectrum matched to ZUFSP Cys-360-labeled peptide sequence. c Pull-down of FLAG-ZUFSP by Ub, SUMO2, or UFM1-conjugated agarose. Lysates from FLAG-ZUFSP-overexpressing cells were enriched on the indicated resins and analyzed by anti-FLAG immunoblotting. d Reactivity of full-length ZUFSP toward ABPs. Purified ZUFSP (wt, C360A, or H491A mutant) was treated with the indicated ABP (Ub-VPS, Ub-VS, Ub-VME, Ub-PA, or SUMO2-VS [S2VS]), then analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. e Reactivity of the catalytic domain of ZUFSP toward ABPs. Purified ZUFSP(298–578) (wt or C360A mutant) was treated with ABP, then analyzed by SDS-PAGE, and stained with Coomassie Brilliant Blue

Fig. 6

ZUFSP is a K63-selective deubiquitinase. a–c Tetra-Ub chain cleavage assays to test activity and selectivity of recombinant full-length ZUFSP a, full-length ZUFSP purified from HEK 293T cells b, and recombinant catalytic domain of ZUFSP c. ZUFSP (1.1 µM) and Ub chains (2.2 µM) were incubated for 1 h at 25 °C, then analyzed by SDS-PAGE and silver staining. d Time-course analysis of ZUFSP-mediated depolymerization of K48- and K63-linked tetra-ubiquitin conjugated to a TAMRA-labeled peptide, monitored by in-gel fluorescence. Figure is representative of two independent replicates. It is noteworthy that this method detects only species bearing a TAMRA fluorophore, and not the distal fragments formed after cleavage, in contrast to silver stains that detect all protein species. e Surface representation of K63-linked di-Ub and K48-linked di-Ub. The color-coding shows residues exhibiting ¹⁵N differential line broadening (DLB) in red and ¹³C DLB in green. Residues were colored if the lines broadened completely (signal disappearance) or the line widths were more than doubled. These DLB effects were observed upon titration of unlabeled catalytically inactive full length ZUFSP^C360A-His₆ to differentially labeled ¹⁵N-proximal ¹³C-distal K48- or K63-linked di-Ubiquitin. K48-linked di-Ubiquitin structure from pdb entry 2kde, K63 di-Ub structure from pdb entry 2rr9

Fig. 7

ZUFSP interacts with and affects the ubiquitination of RPA proteins. a FLAG-immunoprecipitated material from HEK 293T cells expressing wild-type or catalytic mutant FLAG-ZUFSP, or empty vector (EV), was analyzed by western blotting. b HEK 293T cells transfected with siRNA targeting ZUFSP or a non-targeting control were treated with 2 mM hydroxyurea (HU) for 2 h. K63-linked Ub chains were immunoprecipitated and the enriched proteins were analyzed by western blotting