Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi

Abstract

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

Fig 1. Structure of ubiquitin genes in P. chabaudi and comparison of amino acid sequences of the ubiquitin monomer among different organisms.

(A) Organization of the two fusion ubiquitin genes UB_S27a (PCHAS_104020) and UB_L40 (PCHAS_114120); and the polyubiquitin gene (PCHAS_061200). (B) Ubiquitin amino acids sequence alignment from divergent organisms. The identity percentage related to P. chabaudi sequence is shown at the end of each sequence. Changes in the amino acid sequence are underlined in lower case. The conserved lysine residues are shown in bold and indicated with an arrow.

Fig 2. Relative expression of ubiquitin in P. chabaudi.

(A)The mRNA expression of the polyubiquitin gene (PlasmoDB ID PCHAS_0612000) was determined by qRT-PCR and normalized using ribosomal gene 18S as a reference in parasites collected at different time points through the intraerythrocytic cycle (R- Ring, ET Early trophozoite, LT Late trophozoite, ES Early Schizont, and LS Late Schizont). Experiments were performed by duplicates using independent biological samples. (B) Immunofluorescence localization of total ubiquitin conjugates in parasites at their Ring, Trophozoite, or Schizont stages. The parasites were visualized under differential interference contrast (DIC) microscopy. Nuclei were stained with DAPI (blue), and the ubiquitin conjugates were visualized in green. Ubiquitin conjugates are present at all investigated stages.

Fig 3. Immunoblot analyses of ubiquitinated proteins in ring-, trophozoite- and schizont-stage parasites.

Replicate 2-DE gels were transferred onto nitrocellulose membranes and probed with antibodies that recognize mono- and poly-ubiquitinated proteins (panel A) and Lys48-linked polyubiquitin chains (panel B). Top panels correspond to rings, middle panels correspond to trophozoites, and bottom panels correspond to schizonts. Black arrows indicate polypeptides selected for identification by MS/MS (Table 1).

Fig 4. Functional annotation of the Lys48 polyubiquitinated proteins in the three intraerythrocytic stages of P. chabaudi by biological process.

All proteins were detected using anti-Lys48 linked polyubiquitin chains antibodies by 2-DE/IB. Categories were obtained from the Gene Ontology/annotations of biological process at PlasmoDB.