Mapping the protein landscape of the Toxoplasma basal complex

摘要 Apicomplexan寄生虫是人类严重疾病的罪魁祸首。耐药和/或特异性差 不断地破坏治疗这些疾病的治疗方案。为了识别新的药物靶点， P.I.S实验室致力于深化对寄生虫不同细胞生物学过程的理解 从其宿主以弓形虫为模型顶端复合体。在这项提案中，他们将解决这些不同的 结构：基底复合体(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BC的兴趣源于它作为收缩环驱动细胞的功能 组织。基础复合体收缩由一种尚未确定的机制驱动，独立于肌动蛋白- 肌球蛋白使其与宿主区别开来。最近，BC还与其他进程相关联 例如管状泡状空泡内网络(IVN)的组装，它作为 寄生虫和宿主细胞，对确定慢性感染是另外必要的。其他部位的收缩环 系统由125+蛋白质组成，但只有22个BC蛋白质已知。为了破译分子 BC的不同功能背后的机制，建议通过一个在 基于体内邻近的生物素化方法(BioID)。由于BioID提供短距离交互信息 在寄生虫内部的本地建筑群中，BC建筑的专题模型触手可及。为此，一半的人 已知的BC组分将被标记为BioID中的诱饵。已生成的原理实验的证明 一些新的见解强调了这种方法的可行性。定量质谱学数据将被 用于组装蛋白质-蛋白质相互作用(PPI)图，该图有望识别 基础复合体和与许多组件建立连接的节点。预计群集将与 通过(超)结构研究观察到不同的隔室。节点将突出显示潜在的关键组织者 (亚)结构，这使它们成为功能研究的良好目标。为了最大限度地挖掘生物的深度 在这项提案下可以实际实现的见解，10个关键候选人将根据以下情况确定优先顺序 PPI图的位置和生物签名。它们在寄生虫发育过程中的时空动态 BC内的定位将通过自动荧光和/或表位标签进行跟踪。可能发生的动态变化 配合BC的不同组装步骤和/或功能。此外，10名候选人中有5名 将选择代表尽可能多的多样性的主要选择来生成(有条件的) 基因敲除(KO)菌株。将对KO菌株进行BC组装、形态和 收缩以及IVN的形成、形态和功能在宿主细胞养分吸收中的作用。总而言之， 这些数据将有助于解决结构-功能关系，并为分子相互作用和 各种BC功能的基础机制。这些见解将指导未来的机械学研究和 开发特定的新药。

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.