Fig. 4: OTUD5 efficiently cleaves K48 linkages but not K29 linkages.

a OTUD5 efficiently cleaves K11-, K48-, and K63-linked diubiquitin. Diubiquitins (1.0 μg) of eight linkage types were incubated with OTUD5 (0.5 μg) for the indicated duration in an in vitro deubiquitylation assay. The right panel shows quantification of band intensities. b In vitro deubiquitylation assay as in (a) using the indicated tetraubiquitin as substrates. c (i) A schematic workflow for the preparation of K29-Ub4. Enzymatically synthesized Ub4 includes cyclic K29-Ub4 and K29/K48 branched/mixed Ub4 as well as K29-Ub4. Enzymes were used to identify the linkage type of Ub4, and fractions with a high purity of K29-Ub4 were collected. See also online methods. (ii) A schematic workflow for the preparation of K29/K48-Ub8. K29/K48-Ub8 was synthesized from K29-Ub4 and Ub (K48R). The byproduct K29/K48-Ub15~, synthesized by the reaction between K29-Ub4, was removed using cation-exchange chromatography. See also online methods. d In vitro deubiquitylation assay. (i, ii, iii) Either K29-linked Ub4, K48-linked Ub4, or K29/K48-linked Ub8 (1.0 μg) was incubated with OTUD5 (0.5 μg) for the indicated duration. In (iv), the sample of K29/K48-linked Ub8 incubated with OTUD5 for 4 h was stained with anti–K48-linkage antibody. (v), possible architectures of Ub5 in (iv). e In vitro deubiquitylation assay as in (d) with the indicated architectures of Ub2 or Ub3. The graph (iii) shows quantification of four independent experiments (Fig. 4e (i), (ii), and supplementary Fig. 4c). Two-sided ANOVA was used. Source data are provided as a Source Data file.