Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR8204 - CIIL - Center for Infection and Immunity of Lille, Lille, France.
Proteomics platform 3P5-Necker, Université Paris Descartes - Structure Fédérative de Recherche Necker, INSERM US24/CNRS UMS3633, Paris, France.
PLoS Pathog. 2019 Jul 26;15(7):e1007973. doi: 10.1371/journal.ppat.1007973. eCollection 2019 Jul.
The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle.
蛋白磷酸酶 1（PP1）催化亚基在真核生物中的基本和独特功能完全是通过其与众多调节伙伴的相互作用来实现的。在这项工作中，我们报告了配子外蛋白 15（GEXP15）作为 PP1 调节剂的分子和功能特征，GEXP15 是一种疟原虫特异性蛋白。体外相互作用研究表明，GEXP15 通过 P. berghei 中的 RVxF 结合基序与 PP1 发生物理相互作用。功能测定表明，GEXP15 能够增加 PP1 的活性，而 RVxF 基序的突变完全消除了这种调节。P. berghei 中标记的 GEXP15 或 PP1 的免疫沉淀测定，随后进行免疫印迹或质谱分析，证实了它们的相互作用，并表明它们存在于涉及剪接体和蛋白酶体途径的裂殖体和配子体阶段的共享蛋白复合物中，并且在寄生虫发育中发挥重要作用。可行的 GEXP15 缺乏 P. berghei 血寄生虫的表型分析表明，它们无法在 BALB/c 小鼠中引起致死性感染，也无法在 C57BL/6 小鼠中建立实验性脑疟疾。此外，尽管缺乏寄生虫产生配子，但它们没有产生任何卵囊/子孢子，这表明在蚊子中存在高适应成本。GEXP15 缺乏裂殖体的全蛋白组学和磷酸化蛋白质组学分析显示出明显的缺陷，涉及基因表达或入侵调节的蛋白质的丰度和磷酸化状态显著降低。此外，GEXP15 的耗竭似乎主要影响雌性配子体的一些特定蛋白质的丰度。我们的研究首次深入了解了 PP1 调节剂对疟原虫毒力的贡献，并表明 GEXP15 影响无性和有性生命周期。
