Figure 5

TgOTU7 is a deubiquitinase. A Schematic structure of TgOTU7, highlighting the OTU domain. The sequences of the amino acid (aa) sequences of TgOTU7 (TGGT1_271070), HsOTUD6A (NP_056022.1), HsOTUB1 (NP_060140.2), HsOTUD6B (NP_057107.4), and HsOTUB2 (NM_023112.4) are presented. Critical aspartate, cysteine, and histidine residues forming the catalytic triad are highlighted with red stars. B Deubiquitinase activity of TgOTU7. The ubiquitination level of exogenous ubiquitin proteins was measured by Western blotting using individual antibodies against different antigens. Cellular lysates of HEK293T cells transfected with the pRK5-HA-Ub and pRK5-TgOTU7-FLAG plasmids were used. The anti-HA band indicates ubiquitination, and the anti-FLAG band represents TgOTU7. β-Tubulin served as a loading control. The concentrations of the pRK5-TgOTU7-FLAG plasmid increased from left to right, as indicated. These antibodies were also used in Panels C and D. C The critical catalytic domain of TgOTU7 is located between amino acids 149 and 285 (aa). DNA fragments encoding three different regions (1–142 aa, 143–347 aa, 149–285 aa) of TgOTU7 were individually inserted into the pRK5 plasmid to generate mutant TgOTU7 plasmids, which were cotransfected into 293 T cells along with the pRK5-HA-Ub plasmid as labelled. D TgOTU7 shows no linkage specificity in its deubiquitinating activity. A fixed amount of recombinant TgOTU7 plasmid was cotransfected into 293 T cells with plasmids containing different lysine-linked Ub chains (K6, K11, K27, K29, K33, K48, and K63). The first lane in each pair contained the linkage-specific substrate as a control, while the second lane contained recombinant TgOTU7 that exhibited deubiquitinating activity, if present.