Fig. 3: USP53 shows K63-linkage-directed deubiquitination activity.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a,b, Cleavage assay with isopeptide-linked, K63-linked monoubiquitinated or polyubiquitinated substrates. GFP–ubiquitin substrates (1 µM) were incubated with USP5320–383 (500 nM; a) or USP5421–369 (300 nM; b). In-gel fluorescence was used to visualize GFP species, while Coomassie staining of the same gel was used to visualize ubiquitin chains. Arrows indicate cleavage sites. c, USP53 depletion analysis. CaCo-2 cells stably transduced as indicated were analyzed by western blotting after treatment with doxycycline for 72 h. d, Schematic illustration of the diglycine ubiquitinome profiling to identify USP53 substrates. e, Volcano plot showing log2 fold changes of diglycine (ubiquitinated) peptides upon depletion of USP53 in CaCo-2 cells. Proteins linked to phenotypes similar to USP53 mutations are indicated in red. Diglycine sites were unambiguously identified. f, Model linking USP53-associated phenotypes to USP53-modulated ubiquitination of tricellular tight junction proteins (with domains, ubiquitination sites and residue numbers given). PM, plasma membrane. g, MARVELD2 ubiquitination analysis through OtUBD pulldown. CaCo-2 shRNA5 (USP53) cells were treated as in c, lysates were enriched with the high-affinity ubiquitin binder OtUBD and samples were analyzed by western blotting. The triangle highlights MARVELD2 modified with two ubiquitin moieties, emerging upon USP53 depletion. h, MARVELD2 polyubiquitination analysis with K63-specific tUIMRap80 (Rx3A7) TUBE pulldown. i, Analysis of protein levels for samples processed in h. j. Illustration of the workflow used to generate eluates of OtUBD pulldowns for UbiCRest assays. Acidic conditions allowed the elution of ubiquitinated proteins, with the biotinylated OtUBD reagent being retained on streptavidin beads. k, UbiCRest assay. OtUBD eluates prepared as described in j were treated with the indicated DUBs for 1 h at 37 °C and analyzed by western blot. l, UbiCRest assay as in k with USP5320–383 (0.5, 2 and 5 µM) and USP5421–369 (0.3, 1 and 3 µM), showing en bloc deubiquitination of cellular MARVELD2 by recombinant USP53. The results after 2-h incubation are shown in Extended Data Fig. 4k.
