Figure 7.

Role of synthetic peptides derived from PfI2. A. Schematic representation of PfI2 sequence and P1 and P4 peptides derived, respectively, from the RVxF (¹¹KKTISW¹⁶) and HYNE (¹⁰²HYNE¹⁰⁵) domains. P6 (penetrating peptide) and P10 (P1 mutated version) were used as negative controls. B. Interaction studies of P1, P4, P6, P10 with His6-PfPP1 in vitro using ELISA based quantification of binding capacity. Increasing quantities of biotinylated His6-PfPP1 were added to wells coated with peptides (5 μg/well) or recombinant PfI2WT protein (5 μg/well) used as positive control. Results are representative of 4 independent experiments. C. Effect of P1, P4, P6 and P10 on PfPP1 phosphatase activity. Recombinant His6-PfPP1 at 27.03 nM (2.5 μg) was pre-incubated for 30 min at 37°C with different concentrations of peptides or PfI2WT (positive control) before the addition of pNPP. Results presented as % of relative increase or decrease are means ± SEM of four independent experiments performed in duplicate. D. Initiation of G2/M in Xenopus oocytes by peptides. Appearance of GVBD was monitored for 15 h after injection, and values are presented as percentages. Each experiment was performed using a set of 20 oocytes and was repeated at least three times. PfI2WT injection was used as positive control. E. Percentage of GVBD induced by the pre-injection of P1, P4, P6 or P10 (100 ng) 1 hour before PfI2WT injection (100 ng). PfI2WT injection was used as a positive control. F. Abolition of PfI2WT/XePP1 interaction after P1 and P4 peptides pre-injection. Extracts from oocytes pre-injected or not with peptides followed by PfI2WT injection were immunoprecipitated with anti-His mAb. Immunoblot analysis revealed with specific anti-XePP1 antibodies revealed the presence of XePP1 in the complex in P6 and P10 pre-injected extracts (lanes 4 and 5) and in non-pre-injected extracts (lane 1) but not in the P1 and P4 pre-injected extracts (lanes 2 and 3).