Protein phosphatase PfPPM2 signaling and dephosphorylation of Heterochromatin HP1 is critical for asexual division and sexual differentiation of malaria parasite.

We demonstrate that Protein Phosphatase PfPPM2 regulates both asexual and sexual development of human malaria parasite P. falciparum. It is involved in the division of the parasite during asexual development and promotes its sexual differentiation. Phosphoproteomics studies revealed that PfPPM2 targets in the parasite include key regulators of protein translation and chromatin remodelling. We demonstrate that it regulates dephosphorylation of S33 of Heterochromatin Protein 1 (HP1), a regulator of heritable gene silencing and contributes to both mitotic proliferation as well as sexual differentiation of the parasite into sexual forms. Detailed investigations revealed that PfPPM2 mediated dephosphorylation of HP1 at S33 is a signal for parasite sexual conversion. HP1- S33 dephosphorylation impairs its interaction with trimethylated form of histone 3 (H3) at lysine 9 (H3K9me3), which is known to stabilize the heterochromatin. As a result, the expression of transcription of ap2g-which is repressed by HP1-H3K9me3-was stimulated resulting in sexual conversion of the parasite. Interestingly, the state of HP1-S33 phosphorylation seems to largely reflect the fate of the parasite-its optimal phosphorylation is important for asexual division whereas it is dephosphorylated form favours sexual differentiation. PfPPM2 also regulates protein synthesis in the parasite by repressing a signaling pathway involved in the phosphorylation of initiation factor eIF2α, which are likely to contribute to parasite division and possibly differentiation. PfPPM2-HA-glmS:Nf54 parasites were either left untreated or treated with GlcN (cycle 0, rings) and parasite pellets were prepared (schizonts, cycle 1) and RNAseq analysis was perform to compare global changes in transcriptome of these parasites upon PfPPM2 depletion.

我们的研究证实，蛋白磷酸酶PfPPM2（Protein Phosphatase PfPPM2）可调控人类疟疾寄生虫恶性疟原虫（Plasmodium falciparum, P. falciparum）的无性与有性发育过程。该酶参与寄生虫无性发育阶段的增殖分裂，并可促进其向有性阶段分化。磷酸化蛋白质组学（phosphoproteomics）研究显示，寄生虫体内PfPPM2的靶标蛋白涵盖蛋白质翻译与染色质重塑的关键调控因子。我们进一步证实，PfPPM2可调控异染色质蛋白1（Heterochromatin Protein 1, HP1）S33位点的去磷酸化修饰——该位点是可遗传基因沉默的调控位点，其修饰状态同时参与寄生虫的有丝分裂增殖与向有性形式的分化过程。深入研究表明，PfPPM2介导的HP1 S33位点去磷酸化是寄生虫向有性阶段转化的核心信号。HP1 S33去磷酸化会削弱其与组蛋白H3赖氨酸9三甲基化修饰（trimethylated form of histone 3 [H3] at lysine 9, H3K9me3）的结合能力，而H3K9me3已知可稳定异染色质结构。由此，被HP1-H3K9me3复合物抑制的ap2g基因转录水平得以上调，最终推动寄生虫向有性阶段转化。有趣的是，HP1 S33的磷酸化状态似乎在很大程度上决定了寄生虫的发育命运：其最佳磷酸化水平对无性分裂至关重要，而去磷酸化形式则更有利于寄生虫的有性分化。PfPPM2还可通过抑制起始因子eIF2α（initiation factor eIF2α）磷酸化相关的信号通路，调控寄生虫体内的蛋白质合成，这可能对寄生虫的增殖与分化均有贡献。本研究将PfPPM2-HA-glmS:Nf54工程疟原虫分为两组，一组不予处理，另一组以氨基葡萄糖（GlcN）处理（处理时机为第0周期环状体阶段）；随后收集第1周期裂殖体阶段的寄生虫沉淀物，通过RNA测序（RNA-seq）分析，比较PfPPM2敲低后两组寄生虫转录组的全局变化。