Abstract
Malaria is one of the deadliest infectious diseases worldwide. The most severe form is caused by the eukaryotic protozoan parasite Plasmodium falciparum. Recent studies have highlighted the importance of post-translational regulations for the parasite's progression throughout its life cycle, protein ubiquitylation being certainly one of the most abundant. The specificity of its components and the wide range of biological processes in which it is involved make the ubiquitylation pathway a promising source of suitable targets for anti-malarial drug development. Here, we combined immunofluorescent microscopy, biochemical assays, in silico prediction, and mass spectrometry analysis using the multidimensional protein identification technology, or MudPIT, to describe the P. falciparum ubiquitome. We found that ubiquitin conjugates are detected at every morphological stage of the parasite erythrocytic cycle. Furthermore, we detected that more than half of the parasite's proteome represents possible targets for ubiquitylation, especially proteins found to be present at the most replicative stage of the asexual cycle, the trophozoite stage. A large proportion of ubiquitin conjugates were also detected at the schizont stage, consistent with a cell activity slowdown to prepare for merozoite differentiation and invasion. Finally, for the first time in the human malaria parasite, our results strongly indicate the presence of heterologous mixed conjugations, SUMO/UB. This discovery suggests that sumoylated proteins may be regulated by ubiquitylation in P. falciparum. Altogether, our results present the first stepping stone toward a better understanding of ubiquitylation and its role(s) in the biology of the human malaria parasite.
Highlights
Tance of post-translational regulations for the parasite’s progression throughout its life cycle
Ubiquitylation in P. falciparum Erythrocytic Stages—The P. falciparum genome contains more than 100 genes that are potentially encoding the components of the ubiquitylation system, including four sources of ubiquitin (PFL0585w, PF14_0027, PF13_0346, and PF08_0067), eight E1 enzymes, 14 E2 enzymes, more than 50 E3 ubiquitin ligases, and about 30 deubiquitinases [10]
We further investigated the localization of ubiquitin conjugates in intraerythrocytic parasites by immunofluorescence microscopy using an anti-ubiquitin antibody that does not react with free ubiquitin
Summary
Parasite Strain and Culture Conditions—Sorbitol-synchronized P. falciparum parasite strain 3D7 was cultured in human erythrocytes according to previously described protocols [22,23,24]. Parasites were extracted with 0.15% (w/v) saponin (in PBS) for 15 min on ice. Cells were pelleted by centrifugation for 10 min at 3200 ϫ g and 4 °C and washed with PBS (supplemented with NEM, PMSF, and anti-protease mixture) until the supernatant came out clear. Cultures were harvested by 5 min of centrifugation at 800 ϫ g and 4 °C, followed by three washes in PBS supplemented with NEM (20 mM), PMSF (2 mM), and complete mini EDTA-free protease inhibitor mixture (Roche Applied Science). Parasites were extracted with 0.15% (w/v) saponin for 15 min on ice. Cells were pelleted by centrifugation for 10 min at 3200 ϫ g and 4 °C and washed with PBS supplemented with NEM, PMSF, and anti-protease mixture until the supernatant came out clear. STN ratios against control as a base line were calculated using observed mean values and PLGEM-calculated S.D. values as follows
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