Fig. 1: USP53 and USP54 are active DUBs for K63-linked polyubiquitin.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a, Proteomics analysis of ubiquitin–PA probe-labeled proteins in HeLa cell lysate. The volcano plot shows the enrichment (fold change) of proteins detected in HA pulldowns from lysate treated with HA–ubiquitin–PA probe in comparison to HA–UFM1–PA as a control. Identified proteins are shown as dots, HECT E3 ligases are shown in yellow, DUBs are shown in red and USP54 is shown in blue. b, Human DUB families are shown as boxes with numbers of members given in brackets. USP53 and USP54 are annotated as inactive and comprise the most distantly related catalytic domains within the USP family, with highest similarity to the deSUMOylase USPL1. c, Domain architectures of human full-length (FL) and catalytic domain (CD) constructs of USP53 and USP54 used in this study. d, Probe reactivity assay with recombinant catalytic domains. HA–ubiquitin–PA was incubated with wild-type (WT) USP5320–383 and USP5421–369 or inactive mutants of USP5320–383 and USP5421–385. Probe reactivity was analyzed by SDS–PAGE and Coomassie staining. e, Ubiquitin and Ubl RhoG cleavage assay. USP5320–383 (200 nM, top) or USP5421–369 (200 nM, bottom) was added to ubiquitin–RhoG (Ub–RhoG), SUMO1–RhoG (S1–RhoG), SUMO2–RhoG (S2–RhoG), NEDD8–RhoG or ISG15–RhoG (I15–RhoG) and the fluorescence was recorded. Data are shown as the average of technical triplicates. f, Gel-based ubiquitin chain cleavage assay. Specifically linked tetraubiquitin chains (2 µM) were incubated with USP5320–383 (3 µM) or USP5421–369 (300 nM). Samples were taken after the indicated time points and cleavage activity was analyzed by SDS–PAGE and Coomassie staining. diUb, diubiquitin; triUb, triubiquitin; tetraUb, tetraubiquitin.
