Unravelling the function of GEXP15, a regulator of Protein Phosphatase type 1, in Plasmodium falciparum

The Protein Phosphatase type 1 catalytic subunit (PP1c) works in combination with an array of regulatory proteins to specifically dephosphorylate a diverse range of substrates in the cell. In the human malaria parasite, Plasmodium falciparum, this phosphatase and its regulators are poorly characterized. In this study, we employed a multi-faceted approach to investigate the structural and functional characteristics of a conserved Plasmodium specific regulator known as Gametocyte EXported Protein 15, GEXP15. In-silico study highlights three significant regions of interest, an PP1-interacting RVxF motif located in its N-terminal region, a conserved domain whose function is unknown, and a GYF-like domain that may mediate specific protein-protein interactions. In vitro interaction studies shows that GEXP15 has a direct interaction with PP1 via the RVxF binding motif, enhancing its phosphatase activity. Using transgenic GEXP15-tagged line, confocal microscopy reveals that GEXP15 is highly expressed in late asexual stages and is localized in the parasite nucleus. Immunoprecipitation assays followed by mass spectrometry analyses shows its interaction with ribosomal and RNA binding proteins. Furthermore, pulldown analyses of His-tagged recombinant functional domains of GEXP15 confirm its binding to the ribosomal complex via the GYF domain. Our study provides the first insight into the PfGEXP15-PP1-ribosome interaction, crucial for protein translation, and supports PfGEXP15 as a potential target for malaria drug development.

1型蛋白磷酸酶催化亚基（Protein Phosphatase type 1 catalytic subunit，PP1c）可与一系列调控蛋白协同作用，特异性地对细胞内多种底物进行去磷酸化修饰。在人类疟疾寄生虫恶性疟原虫（Plasmodium falciparum）中，该磷酸酶及其调控蛋白的特征研究尚浅。本研究采用多维度研究手段，对一类保守的疟原虫特异性调控蛋白——配子体输出蛋白15（Gametocyte EXported Protein 15，GEXP15）的结构与功能特征展开探究。计算机模拟分析（in silico）结果显示该蛋白存在三个关键功能区域：位于N端的可与PP1结合的RVxF基序、功能未知的保守结构域，以及可能介导特异性蛋白质相互作用的GYF样结构域。体外实验（in vitro）相互作用研究表明，GEXP15可通过RVxF结合基序与PP1直接结合，并增强其磷酸酶活性。通过构建带有标签的GEXP15转基因虫株，共聚焦显微镜成像结果显示，GEXP15在晚期无性生殖阶段高表达，并定位于疟原虫的细胞核内。免疫共沉淀（Immunoprecipitation）实验结合质谱分析（mass spectrometry）结果显示，GEXP15可与核糖体蛋白及RNA结合蛋白发生相互作用。此外，针对带有His标签的GEXP15重组功能结构域的蛋白下拉分析证实，GEXP15可通过GYF结构域与核糖体复合物结合。本研究首次揭示了PfGEXP15-PP1-核糖体相互作用，该相互作用对蛋白质翻译过程至关重要，并证实PfGEXP15可作为疟疾药物开发的潜在靶点。