The unconventional Ark3 cluster in Toxoplasma gondii

弓形虫中非常规的 Ark3 簇

• 批准号: 10511468
• 负责人: Klemens Engelberg
• 金额: $ 23.48万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-20 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10511468
• 关键词: AIDS/HIV problem; Affect; Antibodies; Apical; Apicomplexa; Appearance; Biotin; Biotinylation; Brain; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Line; Cell Nucleus; Cell division; Cell membrane; Cells; Centrosome; Complement; Complex; Cytokinesis; Cytoskeleton; Cytosol; Daughter; Deposition; Distal; Encephalitis; Ensure; Erythrocytes; Exhibits; Eye; Fiber; Future; Genes; Goals; Growth; Heart; Human; Hypersensitivity; Inflammation; Inherited; Knowledge; Lead; Lesion; Ligase; Lytic; Lytic Phase; Maps; Membrane; Microscopy; Microtubules; Mitosis; Molecular; Mothers; Myocarditis; Names; Nature; Nuclear; Nuclear Envelope; Ocular Toxoplasmosis; Opportunistic Infections; Organism; Orthologous Gene; PAWR protein; Parasites; Pathology; Patients; Pharmacotherapy; Phenotype; Phosphotransferases; Plasmodium falciparum; Ploidies; Positioning Attribute; Process; Reporting; Research; Research Proposals; Resolution; Role; Set protein; Side; Signal Transduction; Signaling Molecule; Structure; Testing; Time; Tissues; Toxoplasma gondii; Toxoplasmosis; aurora kinase; aurora kinase A; base; cell assembly; constriction; daughter cell; drug discovery; experimental study; fitness; interest; novel; nuclear division; obligate intracellular parasite; protein degradation; scaffold; segregation; side effect; unborn child

Summary The obligate intracellular parasite Toxoplasma gondii can cause severe opportunistic infection in humans, specially targeting the unborn child or HIV/AIDS patients. Fast rounds of host cell invasion and intracellular replication are responsible for tissue lesions observed in the parasite’s lytic cycle and caused by the tachyzoite form. Although specific drug treatments to battle Toxoplasmosis are available, severe side effects can occur and long-term application might lead to hypersensitivity. Underlying the lytic cycle is a parasite-specific replication apparatus, which forms two new daughter cells in the cytosol of the mother (endodyogeny). This process is highly regulated by parasite-specific kinases, which largely control the cell cycle. Three aurora-related kinases (Ark1-3) have previously been identified and characterized. Among these kinases, Ark3 depletion strongly affects growth of the forming daughters, but detailed actions of the kinase remain unknown. Intriguingly, the kinase has a unique subcellular localization, forming a linear structure during the division process, which extends from the duplicated centrosomes distal towards the nucleus. A similar assembly has also been reported for Plasmodium falciparum, where a similar PfArk3 dynamic is detected towards merozoite formation at the end of the cell cycle in the red blood cell. The nature of this enigmatic assembly has not further been investigated in either organism. The main goal of this proposal is to dissect the nature of the Ark3-related cluster (AC) in the parasite. Catering to this goal is our recent proximity biotinylation-based identification of two new genes whose products exhibit Ark3 dynamics during the cell division. We have named them Ark3-related component (ARC) 1 and 2. Most notably, these hypothetical proteins display severe fitness scores and support the premise that the Ark3/ARC1/2 assembly is executing an essential function at a hereto unrecognized, and completely uncharacterized division structure. We will study the AC by the following two specific aims. Under Aim1 we will revisit the Ark3 phenotype in more detail by conditional protein degradation of the kinase and compare its phenotypic effects to parasites conditionally-depleted of ARC1 and ARC2. Application of ultrastructure expansion microscopy (U-ExM) will further allow us to investigate phenotypic effects and functionally define this mysterious structure. To further map the function, under Aim2 we will reciprocally apply proximity biotinylation to ARC1 and ARC2, using the fast acting biotin ligase TurboID. Upon successful completion of this research proposal we will have gained deep understanding of Ark3 and its unconventional cluster and generate novel insides into early steps of T. gondii daughter cell formation.

概括 专性细胞内寄生虫弓形虫可引起人类严重的机会性感染， 专门针对未出生的婴儿或艾滋病毒/艾滋病患者。 复制负责在寄生虫的裂解周期中观察到的组织损伤，并由 尽管可以使用特定的药物治疗来对抗弓形体病，但其副作用非常严重。 可能会发生影响，长期使用可能会导致过敏。 裂解循环的基础是寄生虫特异性的复制装置，它在细胞中形成两个新的子细胞。 母亲的细胞质（内生）这个过程受到寄生虫特异性激酶的高度调节， 先前已鉴定出三种极光相关激酶 (Ark1-3)。 在这些激酶中，Ark3 缺失强烈影响正在形成的子体的生长， 但该激酶的详细作用仍不清楚。有趣的是，该激酶具有独特的亚细胞。 定位，在划分过程中形成线性结构，该结构从重复的 疟原虫的中心体也有类似的组装情况。 恶性疟原虫，在细胞末端的裂殖子形成中检测到类似的 PfArk3 动态 红细胞中这种神秘组装的性质尚未得到进一步研究。 生物。 该提案的主要目标是剖析寄生虫中与 Ark3 相关的簇（AC）的性质。 为了满足这一目标，我们最近基于邻近生物素化鉴定了两个新基因，它们 产品在细胞分裂过程中表现出 Ark3 动力学，我们将它们命名为 Ark3 相关组件。 (ARC) 1 和 2。最值得注意的是，这些假设的蛋白质显示出严重的健康分数并支持 前提是 Ark3/ARC1/2 组件正在以迄今为止未被识别的方式执行基本功能，并且 我们将通过以下两个具体目标来研究AC。 在 Aim1 下，我们将通过条件蛋白质降解更详细地重新审视 Ark3 表型。 激酶并比较其与条件性去除 ARC1 和 ARC2 的寄生虫的表型效应。 超微结构扩展显微镜（U-ExM）的应用将进一步使我们能够研究表型 效果并在功能上定义这个神秘的结构为了进一步映射该功能，我们将在 Aim2 下。 使用快速作用的生物素连接酶 TurboID，对 ARC1 和 ARC2 相互应用邻近生物素化。 成功完成本研究计划后，我们将对方舟3和 它的非常规簇并在弓形虫子细胞形成的早期步骤中产生新的内部结构。