A unique protein phosphatase with kelch-like domains (PPKL) in Plasmodium modulates ookinete differentiation, motility and invasion.
A unique protein phosphatase with kelch-like domains (PPKL) in Plasmodium modulates ookinete differentiation, motility and invasion.
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疟原虫中具有 kelch 样结构域 (PPKL) 的独特蛋白磷酸酶可调节动动体分化、运动和侵袭。
DOI：
10.1371/journal.ppat.1002948
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发表时间：
2012-09
期刊：
影响因子：
6.7
通讯作者：
Tewari R
中科院分区：
文献类型：
--
作者：
Guttery DS;Poulin B;Ferguson DJ;Szöőr B;Wickstead B;Carroll PL;Ramakrishnan C;Brady D;Patzewitz EM;Straschil U;Solyakov L;Green JL;Sinden RE;Tobin AB;Holder AA;Tewari R
蛋白质的磷酸化和去磷酸化（分别由激酶和磷酸酶催化）是翻译后修饰，在许多真核生物信号通路中起关键作用，并且在人类的许多病理状况中常常失调。在疟原虫中，对其激酶组的功能性了解直到最近才得以实现，其中超过一半的激酶对血液阶段的发育至关重要，另外14种激酶对有性发育和蚊子传播至关重要。然而，任何疟原虫蛋白质磷酸酶的功能都是未知的。在此，我们在啮齿类疟疾模型伯氏疟原虫中利用反向遗传学来研究一种含有kelch样结构域的独特蛋白质磷酸酶（称为PPKL）的作用，该磷酸酶来自一个与拟南芥BSU1相关的家族。系统发育分析证实，包括PPKL在内的BSU1样蛋白家族在陆地植物、绿藻和囊泡虫类的基因组中编码，但在其他真核生物谱系中没有。此外，观察到PPKL属于一个独特的家族，与最密切相关的磷酸酶家族PP1不同。在我们的遗传学方法中，PPKL与C末端绿色荧光蛋白（GFP）融合显示出一种在雌性配子体和动合子中优先表达的活性蛋白质磷酸酶。内源性ppkl基因的缺失导致动合子发育和分化异常，并使顶体微管从内膜复合体解离，产生一种不能运动的表型以及无法侵入蚊子中肠上皮。通过间接免疫荧光评估，这些观察结果通过细胞骨架微管蛋白和肌动蛋白以及敲除突变体中微线体蛋白CTRP的定位变化得到证实。最后，在ppkl - 突变体中观察到对合子发育至关重要的RNA解旋酶dozi的mRNA表达增加，对受精后1.5 - 24小时动合子分化的整体磷酸化研究表明在合子发育的最初几个小时有重大变化。我们的工作证明了独特的PPKL酶在特定阶段的必要性，它调节寄生虫的分化、运动和传播。
疟原虫是单细胞生物，其生命周期在脊椎动物和蚊子宿主之间交替。在蚊子中，疟原虫进行有性发育，即雄性和雌性配子融合形成合子。这个合子然后伸长成为一个侵入性阶段，称为动合子，它能够滑动并穿透蚊子的肠壁以形成一个囊（称为卵囊）。已知蛋白质磷酸化在这个过程中起着至关重要的作用；然而，疟原虫激酶（使蛋白质磷酸化）在合子/动合子成熟过程中的作用比完全未被表征的疟原虫磷酸酶（使蛋白质去磷酸化）的作用被了解得更清楚。利用一种感染小鼠的疟原虫——伯氏疟原虫，我们表明一种含有kelch样结构域的独特蛋白质磷酸酶（称为PPKL）在动合子成熟和运动中起着至关重要作用。删除这个基因会产生形状严重异常的动合子，导致不能运动的寄生虫无法穿透蚊子肠壁的内衬。总体而言，PPKL是一种对动合子发育、运动和侵入至关重要的必需磷酸酶。
Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl− mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission. Malaria parasites are single-celled organisms, which alternate their life-cycle between vertebrate and mosquito hosts. In the mosquito, the malaria parasite undergoes sexual development, whereby a male and female gamete fuse to form a zygote. This zygote then elongates into an invasive stage, termed an ookinete, which can glide to and penetrate the mosquito's gut wall in order to form a cyst (called an oocyst). Protein phosphorylation is known to play a vital role during this process; however, the role of Plasmodium kinases (which phosphorylate proteins) during zygote/ookinete maturation is better understood than the completely uncharacterised plasmodial phosphatases (which dephosphorylate proteins). Using a malaria parasite which infects mice, Plasmodium berghei, we show that a unique protein phosphatase containing kelch-like domains (called PPKL) plays a vital role in ookinete maturation and motility. Deleting this gene produces ookinetes whose shape is grossly abnormal, resulting in non-motile parasites that cannot penetrate the lining of the mosquito gut wall. Overall, PPKL is an essential phosphatase that is critical to ookinete development, motility and invasion.
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影响因子：
3.6
作者：
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通讯作者：
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6.7
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