Characterizing a Serine-Threonine Phosphatase Essential for Asexual Replication in Plasmodium falciparum

摘要 虽然我们在根除疟疾方面取得了重大进展，但由于以下事实阻碍了进一步的努力 人们对恶性疟原虫的基本细胞生物学知之甚少，恶性疟原虫是疟疾的病原体。在……里面 无性生殖阶段，寄生虫在红细胞中复制并导致疟疾特有的周期性 发烧时，一种称为基底复合体的收缩环结构是复制所必需的，并用于分离 萌芽中的子细胞。我已经确定了基本复合体的一个成员，它对寄生虫的生长和 PfPPPP8，作为丝氨酸-苏氨酸磷酸酶。PfPPP8是唯一一种具有 已知的酶活性，我相信PfPPP8对于推动基础的形成和组织是必不可少的 在无性分裂期间很复杂。为了确定它的磷酸酶活性是否使PfPPP8必不可少，我的目标是 利用基因互补。我将使用CRISPR-Cas9插入带有突变的PfPPP8的外源拷贝 在预测的关键催化残基中，进入PfPPP8可诱导的击倒线，击倒内源拷贝， 并确定每个突变对寄生虫生长和分裂的影响。关于PfPPP8‘S’的进一步研究 在分裂过程中，我将在相同的可诱导基因敲除系和 鉴定PfPPP8‘S酶底物。这些底物本身将被基因改造成一种 表位标签和敲除系统，以便可以可视化它们在整个疟原虫分裂过程中的定位 它们缺失的表型后果可以与PfPPP8基因敲除表型进行比较。 为了利用PfPPP8鉴定新的碱基复合蛋白，我对PfPPP8和另一个进行了免疫沉淀 在PfPP8可诱导的敲除背景中，表位标记的基本复合蛋白PfCINCH。我会选择 基于在PfPPP8基因敲除条件下缺乏或耗尽的蛋白质，很可能是 基础复合体的未表征的成员，优先考虑那些被预测为必不可少的成员，以及 引入与假定底物类似的遗传修饰，以确定它们是否也定位于 形成基础复合体所必需的。我还将以同样的方式选择和研究首选候选蛋白质 仅出现在PfPPP8下拉列表中，这些下拉列表可能与PfPPP8具体相关，以便进一步 表征PfPPP8‘S在寄生虫体内的作用机制和整个分裂过程。因为PfPPP8 在致病阶段对疟原虫的生长和复制是必不可少的，因为它只是保守的 在疟原虫物种中，进一步的研究可能证明它是开发抗疟疾药物的有用的新靶点。 面对对现有药物日益增长的抗药性。

Abstract While we have made significant progress toward malaria eradication, further attempts are hampered by the fact that little is known about the basic cellular biology of Plasmodium falciparum, the causative agent of malaria. In the asexual stage, where the parasite replicates in red blood cells and causes malaria’s characteristic cyclical fevers, a contractile ring structure known as the basal complex is required for replication and serves to separate budding daughter cells. I have identified a member of the basal complex that is essential for parasite growth and division, PfPPPP8, and acts as a serine-threonine phosphatase. PfPPP8 is the only basal complex protein with known enzymatic activity, and I believe PfPPP8 is essential to drive the formation and organization of the basal complex during asexual division. To determine whether its phosphatase activity makes PfPPP8 essential, I aim to use genetic complementation. I will use CRISPR-Cas9 to insert an exogenous copy of PfPPP8, with mutations in predicted key catalytic residues, into a PfPPP8 inducible knockdown line, knock down the endogenous copy, and determine the impact of each mutation on parasite growth and division. To further characterize PfPPP8’s role in division, I will perform quantitative phosphoproteomic analysis in the same inducible knockdown line and identify PfPPP8’s enzymatic substrates. These substrates will themselves be genetically modified with an epitope tag and a knockdown system so their localization throughout Plasmodium division can be visualized and the phenotypic consequences of their absence can be compared to the PfPPP8 knockdown phenotype. To identify novel basal complex proteins using PfPPP8, I performed immunoprecipitation on PfPPP8 and another epitope tagged basal complex protein PfCINCH in the PfPPP8 inducible knockdown background. I will select proteins which, based on their absence or depletion in the PfPPP8 knockdown condition, are likely to be uncharacterized members of the basal complex, prioritizing those which are predicted to be essential, and introduce similar genetic modifications as the putative substrates to determine if they also localize to and are required for the formation of the basal complex. I will also similarly select and examine top candidate proteins only present in the PfPPP8 pulldown, which are likely to be specifically relevant to PfPPP8, in order to further characterize PfPPP8’s mechanism of action and role within the parasite throughout division. Because PfPPP8 is essential to Plasmodium growth and replication during the pathogenic stage and because it is conserved only among Plasmodium species, further study could prove it a useful new target for developing antimalarials in the face of rising resistance to extant drugs.