The role of the DOC2.1 protein in Toxoplasma gondii Ca2+- dependent exocytosis

描述（由申请方提供）：顶复门寄生虫弓形虫是免疫功能低下患者危及生命的脑炎的病原体，此外，如果先天感染，还可导致各种出生缺陷。与疾病相关的病理起源于快速的细胞内裂解复制周期。使用遗传方法，我们最近确定了一个DOC 2蛋白（TgDOC 2.1）在钙离子介导的微线体分泌的作用。趋化因子含有成功的宿主细胞侵入和排出所需的粘附分子，这对于完成裂解周期是必不可少的。该提案的目标是阐明TgDOC 2.1在微线体分泌中的作用和功能。这将提供令人兴奋的新的见解到一个知之甚少的机制至关重要的发病机制，不仅弓形虫，但所有其他apicomplexan寄生虫，因为Ca 2+依赖的微线分泌是一个保守的门。与此同时，这将为该途径作为新的特异性药物靶点的潜力提供见解（分泌不是靶点）。 目前批准的药物）。在其他系统中，含有DOC 2结构域的蛋白质募集膜融合机器（例如SNARE和MPEG4蛋白）以促进分泌囊泡与质膜的融合。然而，在TgDOC 2.1中没有与分泌机制相互作用的保守结构域是保守的。TgDOC2.1的比较基因组学鉴定了顶复门中的四个保守序列块，突出了这些结构域的潜在关键作用（块2包含DOC 2结构域）。为了剖析TgDOC 2.1的功能，我们将首先产生试剂，无论是特异性的抗血清或融合报告，以建立其时空亚细胞定位模式整个Ca 2+依赖性胞吐。这将指示TgDOC 2.1在哪个膜发挥其功能。此外，我们将建立一个有条件的TgDOC 2.1敲低的寄生虫系，可用于功能互补的研究与TgDOC 2.1结构域缺失突变体。这将确定哪些结构域在膜易位和/或微线体分泌中起关键作用。在相同的模型中，我们将测试Ca 2+结合结构域中的点突变。Ca 2+结合天冬氨酸残基将首先映射使用一个良好建立的异源哺乳动物组织培养模型。同时，为了了解TgDOC 2.1与哪些蛋白质在分泌过程中合作，我们将使用两种平行方法鉴定与TgDOC 2.1相互作用的蛋白质。第一个是遗传酵母双杂交系统，我们将以TgDOC 2.1为诱饵筛选速殖子cDNA文库。在第二部分中，我们将探索一种名为BioID的新方法。在这种方法中，我们将融合一个E。coliBirA突变体蛋白与TgDOC 2.1重组，并在疟原虫中表达。BirA突变体导致同一复合物中蛋白质的混杂生物素化，随后可以通过链霉亲和素富集和质谱法容易地鉴定。通过任一方法鉴定的推定TgDOC 2.1相互作用伴侣将通过寄生虫中的共定位研究进行验证。总之，我们将定义背后的机制保守的微线体分泌过程。

DESCRIPTION (provided by applicant): The apicomplexan parasite Toxoplasma gondii is the causative agent of life-threatening encephalitis in immunocompromised patients and in addition can cause a variety of birth defects if the infection is contracted congenitally. The pathology associated with disease originates in fast rounds of lytic intracellular replication cycles. Using genetic approach we recently identified a role for a DOC2 protein (TgDOC2.1) in Ca2+- mediated microneme secretion. Micronemes contain adhesion molecules required for successful host cell invasion and egress, which is essential to complete the lytic cycle. The goal of this proposal is to unravel the role and function of TgDOC2.1 in microneme secretion. This will provide exciting new insights into a poorly understood mechanism critical to the pathogenesis of not only Toxoplasma, but to all other apicomplexan parasites since Ca2+-dependent microneme secretion is a conserved across the phylum. At the same time, this will provide insights into the potential of this pathway as a new specific drug target (secretion is not targeted by currently approved drugs). In other systems DOC2-domain containing proteins recruit the membrane fusion machinery (e.g. SNARE and MUNC proteins) to facilitate fusion of the secretory vesicle with the plasma membrane. However, no conserved domains interacting with the secretory machinery are conserved in TgDOC2.1. Comparative genomics of TgDOC2.1 identified four conserved sequence block across the Apicomplexa, highlighting a potentially crucial role for these domains (block 2 contains the DOC2 domain). To dissect TgDOC2.1's function we will first generate reagents, either a specific antiserum or fusion-reporter, to establish its spatio-temporal sub-cellular localization pattern throughout Ca2+-dependent excocytosis. This will indicate at which membrane TgDOC2.1 exerts its function. In addition we will establish a conditional TgDOC2.1 knock-down parasite line that can be used for functional complementation studies with TgDOC2.1 domain deletion mutants. This will identify which domains play a critical role in membrane translocation and/or microneme secretion. In the same model we will test point mutations in the Ca2+-binding domains. The Ca2+- binding Asp residues will first be mapped using a well-established heterologous mammalian tissue culture model. Simultaneously, to learn with which proteins TgDOC2.1 cooperates in the secretion process we will identify proteins interacting with TgDOC2.1 using two parallel approaches. The first is a genetic yeast two- hybrid system wherein we will use TgDOC2.1 as bait to screen a tachyzoite cDNA library. In the second we will explore a new method, named BioID. In this method we will fuse an E. coli BirA mutant protein to TgDOC2.1 and express it in the parasite. The BirA mutant results in promiscuous biotinilyation of proteins in the same complex, which subsequently can be easily identified by streptavidin enrichment and mass spectrometry. Putative TgDOC2.1 interaction partners identified by either method will be validated by co-localization studies in the parasite. Altogether, we will define the mechanism behind the conserved microneme secretion process.