Mapping the protein landscape of the Toxoplasma basal complex

总结 顶复门寄生虫是严重的人类疾病的原因。耐药性和/或特异性差 一直在破坏治疗这些疾病的治疗方案。为了确定新的药物靶点， 私家侦探的实验室专注于加深对寄生虫不同的细胞生物学过程的理解 以弓形虫顶复体为模型，在这份提案中，他们将解决这些不同的问题， 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的兴趣源于其作为收缩环驱动细胞的功能 师.基底复合体收缩是由一种独立于肌动蛋白的尚未定义的机制驱动的- 肌球蛋白使其与宿主分离。最近，《巴塞尔公约》还与其他进程联系在一起 例如管状囊泡内空泡网络（IVN）的组装，其作为交换器在 寄生虫和宿主细胞，并且另外对于建立慢性感染是必需的。收缩环在其他 系统由125+种蛋白质组成，但只有22种BC蛋白质是已知的。去破译 BC的各种功能背后的机制，建议通过一个 体内邻近生物素化方法（BioID）。由于BioID提供短距离交互信息， 在寄生虫体内的原生复合体中，BC架构的局部模型触手可及。为此， 已知的BC组分将在BioID中被标记为诱饵。已生成的原理验证实验 一些新的见解强调了这一办法的可行性。定量质谱数据将 用于组装蛋白质-蛋白质相互作用（PPI）图谱，预计该图谱将识别蛋白质内的两个簇。 基底复合体和与许多组成部分连接的节点。集群预计将与 通过（超）结构研究观察到的不同隔室。节点将突出潜在的关键组织者， （子）结构，这使它们成为功能研究的良好目标。为了最大限度地提高生物学的深度 根据本提案可以切实实现的见解，将根据以下因素优先考虑10名关键候选人： PPI图位置和生物特征。它们在寄生虫发育过程中的时空动态 并且BC内的定位将通过自体荧光和/或表位标签来跟踪。动态变化可能 与BC的不同组装步骤和/或功能保持一致。此外，10名候选人中有5名 代表尽可能多的多样性的主要选择将被选择用于生成（条件） 基因敲除（KO）菌株。将评价KO菌株的BC组装缺陷、形态和 收缩以及IVN的形成、形态和在宿主细胞营养摄取中的功能。总之， 这些数据将有助于解决结构-功能关系，并提供分子相互作用的提示， 各种BC功能的基础机制。这些见解将指导未来的机制研究， 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.