The Toxoplasma basal complex in cell division

细胞分裂中的弓形虫基础复合体

基本信息

批准号：
10552584
负责人：
Marc-Jan Gubbels
金额：
$ 37.53万
依托单位：
BOSTON COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-10 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10552584
关键词：
Actins Adherence Adhesions Affect Alveolar Apical Binding Biological Biotinylation C-terminal Cell Division Process Cell division Cells Centrosome Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Congenital Abnormality Cytoskeleton Data Data Set Daughter Dissection Drug Targeting Encephalitis Enzymes Family Genes Genetic Grant Hypersensitivity Hypothetical Protein In Vitro Intermediate Filament Proteins Kinetics Knock-out Libraries Lytic Phase Maps Membrane Microtubule-Associated Proteins Microtubules Modeling Mothers Motor Myosin ATPase Opportunistic Infections Parasites Parents Pathology Pharmaceutical Preparations Phenocopy Phenotype Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Population Process Protein C Proteins Proteomics Recombinants Regulation Resistance Role Running Scaffolding Protein Signal Transduction Solid Structure Testing Toxoplasma Toxoplasma gondii Toxoplasmosis Transfection Vesicle Xenopus axon guidance candidate identification candidate validation constriction daughter cell factor EF-P fitness foodborne infection genome-wide in vitro testing inorganic phosphate insight knock-down membrane skeleton microtubule-associated protein 1B mutant new therapeutic target overexpression prevent protein complex recruit scaffold screening spatiotemporal

项目摘要

Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. Under this proposal the unique cell division process will be interrogated to identify putative new drug targets. Toxoplasma divides by a distinct internal budding process whereby two daughter parasites are assembled within a mother cell. The cortical membrane skeleton composed of flattened alveolar vesicles supported by an epiplastin protein network and 22 subpellicular microtubules (MTs) is nucleated on the centrosomes and assembles in an apical to basal direction. In the second half of division the posterior end of the daughter buds (i.e. the basal complex or BC) starts to taper driven by Myosin J (MyoJ). Absence of MyoJ only modestly impact parasite viability, even while it leaves the BC somewhat unconstricted, fitting classic data on cell division resistance to actin depolymerizing agents. However, preventing assembly of the BC altogether by depleting or overexpressing the BC scaffolding protein MORN1 results in parasites with fraying MTs unable to complete cell division and has dramatic impact on viability. To unravel this intriguing process, under an R21 grant the BC was proteomically dissected through proximity dependent biotinylation (BioID) on 8 BC components. This revealed 4-5 different protein complexes aligning with the ultrastructure. Two key observations are further pursued under this proposal: 1. A putative MT Associated Protein, MAP1B-L1, appears to assemble on the (+)-ends of the subpellicular MTs and is essential for BC assembly and parasite viability; 2. Several kinases and phosphatases identified indicate the BC is regulated by differential phosphorylation. Under Aim 1 MAP1B-L1 and another critical BC MAP dubbed MAP1B- L2 will be tested for MT binding capacity by generating deletion mutants in the parasite, in vitro using the identified MT binding domains, and by exogenous expression in the Xenopus leavis axon guidance model as relevant to related MAPs. Under Aim 2 we will pursue four additional candidates identified in the BioID approach with a likely essential function, which are all hypothetical proteins narrowly conserved in internally budding parasites and harbor putative adhesion domains. In addition, we will apply fast acting TurboID on BC components transiently associating with the assembling BC like MAP1B-L1 as these were likely undersampled in the current dataset, yet define the essential step of the BC in cell division. Under Aim 3 we will subject 2 kinases and 1 phosphatase to synthetic lethality screening using the genome wide CRISRP/Cas9 library. Preliminary data of the first kinase tested already demonstrates experimental feasibility and revealed interesting new insights. Combining the proteomic and genetic data sets is expected to provide a solid basis to assemble the wiring diagram of the BC. In the current working model the BC is first assembled on the MT (+)-ends, followed by recruitment of adhesion proteins to keep the MT-ends together. Overall, this is expected to deliver exciting new insights into internal budding, how it differs from schizogony, and could highlight new drug targets.

概括弓形虫Gondii是一种强制性的细胞内apicomplexan寄生虫，引起了严重的机会性感染。当前的药物容易引起超敏反应，尤其是长期使用时。根据该提议独特的细胞分裂过程将受到质疑，以识别推定的新药物靶标。弓形虫除以独特的内部发芽过程，将两个女儿寄生虫组装在母细胞中。这皮质膜骨骼由表皮蛋白网络支撑的扁平肺泡囊泡组成和22个亚细胞微管（MTS）在中心体上和基础上的中心体和组装成核。方向。在分区的后半部分，女子芽的后端（即基底复合物或BC）肌球蛋白J（MyoJ）驱动的锥度开始逐渐变细。没有Myoj仅适度影响寄生虫的生存能力使卑诗省有些无关，拟合了有关细胞分裂抗肌动蛋白解聚的经典数据代理商。但是，通过耗尽或过表达BC脚手架，完全防止BC组装蛋白质MORN1导致寄生虫具有磨损MT无法完成细胞分裂的寄生虫，并且具有巨大的影响关于生存能力。为了揭示这个有趣的过程，根据R21授予，卑诗省是通过蛋白质组剖析的公元前8 BC成分的接近依赖性生物素化（生物差）。这揭示了4-5种不同的蛋白质复合物与超微结构对齐。根据该提案进一步提出了两个关键观察：1。推定的MT 相关蛋白Map1b-l1似乎在亚细胞MT的（+） - 末端组装，是必不可少的用于卑诗省的组装和寄生虫的生存能力； 2。鉴定出的几种激酶和磷酸酶表明BC为受差异磷酸化调节。在AIM 1 MAP1B-L1和另一个关键的BC映射配音MAP1B- L2将通过在寄生虫中产生缺失突变体来测试MT结合能力，并在体外使用鉴定出MT结合结构域，并通过在爪蟾Leavis Axon引导模型中通过外源表达为与相关地图有关。在AIM 2下，我们将在BioID方法中攻读其他四名候选人具有可能的必不可少的功能，它们都是假设的蛋白质，在内部发芽中狭义寄生虫和港口推定的粘附域。此外，我们将在卑诗省的组件上应用快速的表演涡轮与MAP1B-L1（MAP1B-L1）的组装瞬时相关数据集，但定义了BC在细胞分裂中的基本步骤。在AIM 3下，我们将对2个激酶和1个使用基因组宽CRISRP/CAS9库进行磷酸酶进行合成致死性筛查。初步数据测试的第一个激酶已经证明了实验性的可行性，并揭示了有趣的新见解。结合蛋白质组学和遗传数据集有望提供扎实的基础来组装布线卑诗省的图。在当前的工作模型中，BC首先组装在MT（+） - 结束上，其次是募集粘附蛋白以保持MT末端。总的来说，这将有望带来令人兴奋的新对内部萌芽的洞察力，与精神分裂的不同，并可以突出新药靶标。