Mapping the protein landscape of the Toxoplasma basal complex

绘制弓形虫基础复合物的蛋白质图谱

• 批准号: 9387832
• 负责人: Marc-Jan Gubbels
• 金额: $ 23.48万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-22 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387832
• 关键词: Actins; Address; Affect; Apical; Architecture; Automobile Driving; Biological; Biological Process; Biotin; Biotinylation; Bypass; Cell Division Process; Cell division; Cells; Centrosome; Chronic; Cluster Analysis; Complex; Comprehension; Congenital Abnormality; Cytoskeleton; Data; Daughter; Defect; Development; Disease; Drug Targeting; Drug resistance; Encephalitis; Epitopes; Fission Yeast; Future; Generations; Genes; Goals; Immune system; In Vitro; Infection; Knock-out; Ligase; Lytic Phase; Maintenance; Mammalian Cell; Mass Spectrum Analysis; Membrane; Modeling; Molecular; Monitor; Morphology; Mothers; Mus; Myosin ATPase; Nature; Nutrient; Parasites; Pathogenesis; Pathology; Pattern; Positioning Attribute; Process; Protein-Protein Interaction Map; Proteins; Proteome; Recruitment Activity; Regimen; Role; Solubility; Specificity; Stratum Basale; Structure; Structure-Activity Relationship; System; Testing; Therapeutic; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vacuole; Work; base; constriction; daughter cell; design; experimental study; foodborne infection; human disease; in vivo; insight; interest; membrane skeleton; mutant; new therapeutic target; next generation; novel; novel therapeutics; protein complex; scaffold; spatiotemporal; stem; uptake

Summary Apicomplexan parasites are responsible for severe human diseases. Drug resistance and/or poor specificity are constantly undermining therapeutic regimens to treat these diseases. In order to identify new drug targets, the P.I.'s lab focuses on deepening the understanding of cell biological processes wherein the parasite differs from its host using Toxoplasma gondii as model apicomplexan. In this proposal they will address such distinct structure: the basal complex (BC), which sits at the posterior end of the unique cortical membrane skeleton of these parasites. Historically, interest in the BC stems from its function as the contractile ring driving cell division. Basal complex contraction is powered by an as yet undefined mechanism independent of actin- myosin setting it apart from the host. More recently, the BC has also been associated with other processes such as assembly of the tubulovesicular intravacuolar network (IVN), which operates as an exchanger between parasite and host cell and is additionally essential to establish a chronic infection. Contractile rings in other systems are composed of 125+ proteins, yet only 22 BC proteins are known. To decipher the molecular mechanisms behind the BC's diverse functions, it is proposed to assemble its complete parts list through an in vivo proximity-based biotinylation approach (BioID). Since BioID provides short-distance interaction information in the native complex inside the parasite, a topical model of BC architecture is within reach. To that end half the known BC components will be tagged as baits in BioID. Proof of principle experiments already generated several new insights underscoring the feasibility of this approach. Quantitative mass spectrometry data will be used to assemble a protein-protein interaction (PPI) map, which is expected to identify both clusters within the basal complex and nodes that make connections with many components. Clusters are expected to align with different compartments observed by (ultra)structural studies. Nodes will highlight potential key organizers of (sub)structures, which makes them good targets for functional studies. To maximize the depth of biological insights that can be realistically achieved under this proposal, 10 key candidates will be prioritized based on PPI map position and biological signature. Their spatiotemporal dynamics throughout parasite development and localization within the BC will be tracked by auto-fluorescent and/or epitope tags. Dynamical changes likely align with different assembly steps and/or functions of the BC. Furthermore, 5 candidates among the 10 primary picks representing as much diversity as possible will be selected for the generation of (conditional) gene knock-out (KO) strains. The KO strains will be evaluated for defects in BC assembly, morphology and constriction as well as IVN formation, morphology and function in uptake of host cell nutrients. Altogether, these data will help to resolve structure-function relationships and provide a hint at molecular interplay and mechanisms underlying the various BC functions. These insights will guide future mechanistic studies and development of specific new drugs.

概括 Apicomplexan寄生虫负责严重的人类疾病。耐药性和/或特异性差 不断破坏治疗治疗这些疾病的治疗方案。为了识别新药物靶标 P.I.的实验室重点是加深对寄生虫不同的细胞生物学过程的理解 从其宿主使用弓形虫作为型号的Apicomplexan。在这个建议中，他们将解决如此独特的 结构：基底复合物（BC），位于独特的皮质膜骨架的后端 这些寄生虫。从历史上看，对卑诗省的兴趣源于其作为收缩环驱动器的功能 分配。基础复杂收缩由尚未定义的机制提供动力，与肌动蛋白无关 肌球蛋白将其与主机区分开。最近，卑诗省也与其他过程相关联 例如小管膜出现弹药网络（IVN）的组装，该网络是在交换器之间运行的 寄生虫和宿主细胞，对于建立慢性感染也是必不可少的。收缩戒指 系统由125多种蛋白质组成，但仅知道22个BC蛋白。破译分子 卑诗省各种功能背后的机制，提议通过AN组装其完整零件清单 基于体内近端的生物素化方法（生物片）。由于Bioid提供短途交互信息 在寄生虫内部的本地复合物中，可以触及卑诗省建筑的局部模型。到那一半 已知的BC组件将被标记为Bioil中的诱饵。已经生成的主要实验证明 一些新的见解强调了这种方法的可行性。定量质谱数据将是 用于组装蛋白质 - 蛋白质相互作用（PPI）图，预计将识别两个簇 基础复合物和与许多组件建立连接的节点。簇有望与 通过（超）结构研究观察到的不同隔室。节点将重点介绍潜在的关键组织者 （子）结构，使其成为功能研究的良好目标。为了最大化生物学的深度 根据该提案可以实际实现的见解，将根据10个关键候选人的优先级 PPI地图位置和生物学签名。他们整个寄生虫发育中的时空动态 BC内的本地化将通过自动荧光和/或表位标签跟踪。动态变化可能 与BC的不同组装步骤和/或功能对齐。此外，10名候选人 将选择代表尽可能多多样性的主要选择（条件） 基因敲除（KO）菌株。 KO菌株将在BC组装，形态和 收缩以及IVN的形成，形态和功能在摄取宿主细胞营养中。共， 这些数据将有助于解决结构功能关系，并提示分子相互作用和 各种BC函数的基础机制。这些见解将指导未来的机械研究和 开发特定的新药。