Figure 4

Characterizations of PfUSP14 as a proteasome-associated DUB.

(A) Interactions of PfUSP14 with plasmodial proteasomes were examined in a GST pull-down assay using P. falciparum crude extracts. Equimolar amount of GST-tagged full-length PfUSP14 (GST-F), the UBL domain of PfUSP14 (GST-UBL), the catalytic part (GST-CP), or GST (control) was used for each pull-down experiment, as indicated by immunoblotting of the bait proteins using an anti-GST antibody. The pulled-down plasmodial proteasomes were resolved by 12% SDS-PAGE and detected by immunoblotting using an antibody recognizing plasmodial 20S α-subunits (Sessler et al., 2012). (B) Robust hydrolysis of Ub-AMC by recombinant PfUSP14. Free hUSP14 was totally inactive in hydrolyzing Ub-AMC under the same experimental conditions. As a control, hUSP14 was activated by the presence of ubiquitin-vinyl sulfone (Ub-VS)-treated human proteasome (26S-VS), which lacks endogenous deubiquitinating activity. (C) Cleavage of Lys48-linked di-ubiquitin (Di-Ub) by recombinant PfUSP14. Omitting a reducing agent dithiothreitol (DTT) in the assay buffer or adding a thiol-blocking agent N-ethylmaleimide (NEM) significantly reduced PfUSP14 activity. Free hUSP14 did not cleave Di-Ub. Ubiquitin species were detected using immunoblotting with an anti-ubiquitin antibody. Coomassie blue staining was used to show the amount of the enzyme (50% input) used in each experiment. (D) The ubiquitin C-terminus hydrolysis activity of PfUSP14 can be enhanced by the presence of purified, Ub-VS-treated plasmodial 26S proteasomes (Pf26S). RFU, relative fluorescence unit. The results of one representative experiment are shown.