Figure 6
From: Characterization of the 26S proteasome network in Plasmodium falciparum
The DUB inhibitor b-AP15 inhibits PfUSP14 and exhibits antimalarial effects on intraerythrocytic P. falciparum.
(A) Concentration-dependent inhibition of PfUSP14 by b-AP15. (B) b-AP15 potently inhibits parasite growth of chloroquine-sensitive (3D7) or chloroquine-resistant (Dd2) P. falciparum strains in a dose-dependent manner. (C) P. falciparum (3D7, about 38 h-old) were treated with b-AP15 of 2-fold IC50 (3.08 μM), 5-fold IC50 (7.7 μM) and 10-fold IC50 (15.4 μM) concentrations for 4 hours. The morphology of P. falciparum parasites was then analyzed in a blood smear where the parasites in red blood cells were stained with Giemsa. (D) Endogenous K48-linked polyubiquitinylated proteins (primary proteasomal substrates) in the b-AP15-treated parasites were resolved in 7.5% SDS-PAGE and analyzed by immunoblotting using a specific antibody recognizing K48-linked ubiquitin conjugates. Equal amounts of parasite extracts were loaded as revealed by Ponceau S staining. Free ubiquitin and the plasmodial proteasomes in the parasites were analyzed in parallel by immunoblotting using respective antibodies. (E) The proteasomal activity and total DUB activity in b-AP15-treated parasite were determined using a Suc-LLVY-AMC hydrolysis assay and a Ub-AMC hydrolysis assay, respectively.
