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

描述恶性疟原虫无性复制所必需的丝氨酸-苏氨酸磷酸酶

• 批准号: 10382628
• 负责人: Alexander Aeneas Morano
• 金额: $ 4.05万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10382628
• 关键词: Antimalarials; Biological; Biological Assay; Biology; CRISPR/Cas technology; Cell Division Process; Cell division; Cells; Cellular biology; Cessation of life; Characteristics; Complement; Complex; Cre lox recombination system; Dangerousness; Data; Defect; Development; Epitopes; Erythrocytes; Failure; Fever; Flow Cytometry; Genes; Genetic; Goals; Growth; Harvest; Human; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Knock-out; Malaria; Modification; Molecular; Mutation; Parasites; Pathogenicity; Periodicity; Pharmaceutical Preparations; Phenocopy; Phenotype; Phosphoric Monoester Hydrolases; Plasmodium; Plasmodium falciparum; Play; Point Mutation; Process; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; Recombinant Proteins; Recombinants; Resistance; Role; Selection Criteria; Set protein; Structure; Subgroup; System; Testing; Transgenic Organisms; asexual; base; daughter cell; fitness; genetic analysis; in vivo; knock-down; member; novel; phosphoproteomics; protein complex; protein protein interaction

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.

摘要 虽然我们在消灭疟疾方面取得了重大进展，但进一步的努力受到以下事实的阻碍： 人们对疟疾病原体恶性疟原虫的基本细胞生物学知之甚少。在 无性阶段，寄生虫在红细胞中复制，导致疟疾的周期性特征， 发烧时，复制需要一种称为基底复合体的收缩环结构， 出芽的子细胞我已经确定了寄生虫生长所必需的基底复合体的一个成员， 分裂，PfPPPP 8，并作为丝氨酸-苏氨酸磷酸酶。PfPPP 8是唯一一种具有 已知的酶活性，我相信PfPPP 8是必不可少的驱动形成和组织的基础 在无性分裂期间复杂。为了确定PfPPP 8的磷酸酶活性是否是必需的， 利用基因互补。我将使用CRISPR-Cas9插入PfPPP 8的外源拷贝， 在预测的关键催化残基中，进入PfPPP 8诱导型敲低系，敲低内源拷贝， 并确定每个突变对寄生虫生长和分裂的影响。为了进一步表征PfPPP 8的 在分裂中的作用，我将在相同的诱导型敲低系中进行定量磷酸蛋白质组学分析， 鉴定PfPPP 8的酶底物。这些底物本身将用一种 表位标签和敲低系统，因此它们在疟原虫分裂过程中的定位可以可视化， 其缺失的表型结果可与PfPPP 8敲低表型进行比较。 为了使用PfPPP 8鉴定新型基础复合物蛋白，我对PfPPP 8和另一种蛋白进行了免疫沉淀 表位标记的基础复合物蛋白PfCINCH在PfPPP 8可诱导敲低背景中的表达。我将选择 蛋白质，基于它们在PfPPP 8敲低条件下的缺失或耗尽，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需的成员，以及 引入类似的遗传修饰作为推定的底物，以确定它们是否也定位于 这是形成基底复合体所必需的。我也将类似地选择和检查顶级候选蛋白质 仅存在于PfPPP 8下拉中，其可能与PfPPP 8具体相关，以便进一步 描述PfPPP 8在整个分裂过程中在寄生虫中的作用机制和作用。因为PfPPP 8 在致病阶段对疟原虫的生长和复制是必需的，因为它仅 在疟原虫物种中，进一步的研究可以证明它是开发抗疟疾药物的一个有用的新靶点， 面对对现存药物的耐药性不断上升。