Fig. 3: Methylation of USP11 at R433 regulates DUB activity.
a Predicted structure of the catalytic core of USP11 using USP4 and USP15 structures as templates (PDB IDs: 2Y6E, 6GHA) showing the location of the distal (S1) ubiquitin-binding site, the catalytic residues (orange) and arginine 433 (red). b In vitro DUB assay using K63-linked di-ubiquitin (K63-diUb) as substrate. 293T cells were depleted of endogenous USP11 and then transfected with the indicated constructs and Flag-tagged USP11 immunoprecipitated. Following Flag peptide elution, immunoprecipitated USP11 was incubated with K63-diUb for time-course analysis of di-ubiquitin chain processing (representative image of n = 3 independent biological repeats). Lanes 1–14 were run on a single gel, dotted line is to aid interpretation. c Depletion of PRMT1 reduces USP11 activity as determined by in vitro DUB assay using K63-linked di-ubiquitin as substrate. Flag-USP11 was immunoprecipitated from 293T Flp-In-Flag-USP11 cells stably depleted for PRMT1 and peptide eluted. Flag-USP11 was incubated with K63-diUb for time-course analysis of di-ubiquitin chain processing (representative image of n = 3 independent biological repeats). All lanes were run on a single gel, the dotted line is to aid interpretation. d Methylation of USP11 alters its DUB activity. Recombinant wild-type or R433K His-tagged USP11 was incubated with GST-PRMT1, SAM (as indicated), and increasing concentrations of Ub-AMC. DUB activity was measured through fluorescence detection of cleaved AMC. Data were fitted according to Michaelis–Menten model using GraphPad Prism. Enzymatic parameters Km, Vmax and catalytic efficiency (kcat/Km) are shown in the table below (mean ± SD; n = 3 independent biological repeats: one-way ANOVA and Tukey post hoc test: Vmax: His-USP11/GST-PRMT1/SAM versus His-USP11-R433K/GST-PRMT1/SAM **p = 0.0035; His-USP11/GST-PRMT1/SAM versus His-USP11/GST-PRMT1 *p = 0.0438; kcat/Km: His-USP11/GST-PRMT1/SAM versus His-USP11-R433K/GST-PRMT1/SAM **p = 0.0034; His-USP11/GST-PRMT1/SAM versus His-USP11/GST-PRMT1 *p = 0.0104. NS not significant. Uncropped blots and processed graphical data provided as a Source Data file.
