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 顶复门寄生虫是造成严重人类疾病的原因。耐药性和/或特异性差 不断破坏治疗这些疾病的治疗方案。为了确定新的药物靶点， P.I. 的实验室致力于加深对寄生虫不同的细胞生物过程的理解 使用弓形虫作为模型 apicomplexan 从其宿主中提取。在这项提案中，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 分配。基础复合体收缩是由一种尚未定义的独立于肌动蛋白的机制提供动力的。 肌球蛋白将其与宿主区分开来。最近，BC 还与其他流程相关联 例如管状水泡内液泡网络 (IVN) 的组装，该网络作为之间的交换器 寄生虫和宿主细胞，并且对于建立慢性感染也是必需的。其他收缩环 系统由 125 多种蛋白质组成，但已知的 BC 蛋白质只有 22 种。破译分子 为了了解 BC 多种功能背后的机制，建议通过内部集成来组装其完整的零件清单。 基于体内邻近性的生物素化方法（BioID）。由于BioID提供短距离交互信息 在寄生虫内部的原生复合体中，BC 架构的局部模型触手可及。为此，一半 已知的 BC 成分将在 BioID 中标记为诱饵。原理实验证明已经生成 一些新的见解强调了这种方法的可行性。定量质谱数据将 用于组装蛋白质-蛋白质相互作用（PPI）图，预计该图可以识别 基底复合体和与许多组件建立连接的节点。集群预计将与 通过（超）结构研究观察到不同的隔室。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。最大化生物深度 根据该提案可以实际实现的见解，将根据以下因素优先考虑 10 名关键候选人 PPI 图谱位置和生物特征。它们在寄生虫发育过程中的时空动态 BC 内的定位将通过自身荧光和/或表位标签进行跟踪。可能发生动态变化 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 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。将评估 KO 菌株的 BC 组装、形态和 收缩以及 IVN 的形成、形态和吸收宿主细胞营养的功能。共， 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和 各种 BC 功能背后的机制。这些见解将指导未来的机制研究和 特定新药的开发。