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 强制性细胞内寄生虫弓形虫弓形虫会引起人类严重的机会感染， 针对未出生的儿童或艾滋病毒/艾滋病患者的特殊目标。宿主细胞入侵和细胞内的快速回合 复制负责在寄生虫的裂解周期中观察到的组织病变，并由 tachyzoite形式。尽管可以使用特定的药物治疗毒性毒素，但严重的一面 可能会发生影响，并且长期应用可能导致过敏。 裂解周期的基础是一种寄生虫特异性复制设备，在 母亲的细胞质（内部发育）。该过程受寄生虫特异性激酶的高度调节， 这在很大程度上控制了细胞周期。先前已经鉴定出三个与Aurora相关的激酶（ARK1-3） 并描述了。在这些激酶中，ARK3加深了强烈影响成立女儿的成长， 但是激酶的详细动作仍然未知。有趣的是，激酶具有独特的亚细胞 本地化，在分裂过程中形成线性结构，该结构从重复的 中心体向核心远端。还报道了类似的组装 恶性，其中在细胞末端检测到类似的PFARK3动态朝向梅罗祖岩形成 在红细胞中循环。该神秘组装的性质尚未进一步研究 生物。 该提案的主要目的是剖析寄生虫中与ARK3相关簇（AC）的性质。 满足这一目标的是我们最近基于生物素化的近端对两个新基因的识别，它们的基因 产品在细胞分裂过程中表现出ARK3动力学。我们将其命名为ARK3相关的组件 （弧）1和2。最值得注意的是，这些假设的蛋白质表现出严格的健身评分，并支持 前提是ARK3/ARC1/2组件正在迄今未认可的基本功能，并且 完全未表征的划分结构。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访问ARK3表型 激酶并将其表型效应与有条件脱落的ARC1和ARC2的寄生虫进行比较。 超微结构扩展显微镜（U-EXM）的应用将进一步允许我们研究表型 影响并在功能上定义了这种神秘的结构。为了进一步映射功能，在AIM2下，我们将 使用快速作用的生物素连接酶涡轮增压，对ARC1和ARC2的亲近生物素化将接近性生物素化施加。 成功完成这项研究建议后，我们将对ARK3和 它的非常规的簇，并产生新颖的群体，内部陷入了弓形虫子细胞形成的早期步骤中。