Extended Data Fig. 1: USP53 and USP54 are active DUBs.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a. Schematic of the workflow used to identify active DUBs. HeLa cell lysate was treated with HA-Ub-PA or HA-UFM1-PA probe. Labelled proteins were enriched and detected by mass spectrometry. b. Schematic of the probe reactivity assay. The C-terminal propargylamine (PA) warhead reacts with the catalytic cysteine of DUBs to a vinyl thioether, forming a covalent DUB~Ub-PA complex. c. Ub/Ubl specificity assayed through probe reactivity with recombinant catalytic domains. A panel of HA-Ub/Ubl-PA probes was incubated with wild-type USP5320-383 or wild-type USP5421-369. Probe reactivity was analyzed by SDS-PAGE and Coomassie staining. Uncropped versions of all gels are provided as Source Data. d. Intact protein mass spectrometry of probes used in panel c. Expected and found masses are given in Dalton. The star denotes an HA-SUMO41–92 species N-terminally truncated by four residues. All other probes have been characterized previously41. e. Schematic of the Ubiquitin-RhodamineG (Ub-RhoG) cleavage assay. DUBs cleave the amide bond between ubiquitin and RhoG and thereby liberate fluorescent RhoG. f. Ub-RhoG cleavage assay. Indicated concentrations of USP5320-383 (left) and USP5421-369 (right) or of the catalytic inactive mutants were added to Ub-RhoG and fluorescence was recorded. Data are shown as average of technical triplicates. CS enzymes denote the use of catalytic cysteine to serine mutated enzymes as in Fig. 1d. Of note, this activity is approximately 3 to 4 orders of magnitude weaker than for other USP DUBs including USP2, USP7, USP36 and USP4241.
