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

DOC2.1蛋白在弓形虫Ca2依赖性胞吐作用中的作用

基本信息

批准号：
8716658
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-09 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8716658
关键词：
Antibodies Apicomplexa Binding Binding Proteins Biological Process C2 Domain Cell Adhesion Molecules Cell membrane Cells Complex Congenital Abnormality Conserved Sequence Contracts Disease Dissection Drug Targeting Encephalitis Escherichia coli Exocytosis Family Genetic Genomics Goals Immune Sera Immunocompromised Host Infection Ionophores Ions Learning Life Light Lytic Lytic Phase Malaria Mammalian Cell Maps Mass Spectrum Analysis Mediating Membrane Membrane Fusion Methods Modeling Names Opportunistic Infections Organelles Parasites Pathogenesis Pathology Pathway interactions Pattern Pharmaceutical Preparations Plasmodium Play Point Mutation Process Protein Family Proteins Reagent Recruitment Activity Reporter Role SNAP receptor Secretory Vesicles Series Signal Pathway Site Streptavidin System Testing Time Tissues Toxoplasma Toxoplasma gondii Vesicle Yeasts cDNA Library cell motility comparative genomics domain mapping insight knock-down lytic replication member mutant neurotransmitter release protein function protein structure public health relevance research study secretion process temperature sensitive mutant tissue culture yeast two hybrid system

项目摘要

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.

描述（由申请人提供）：Apicomplexan寄生虫弓形虫Gondii是免疫功能低下的患者威胁生命的脑炎的病因，如果先天感染签约，可能会导致多种出生缺陷。与疾病相关的病理起源于裂解的细胞内复制周期。使用遗传方法，我们最近确定了Ca2+介导的微元分泌中DOC2蛋白（TGDOC2.1）的作用。微生物包含成功的宿主细胞侵袭和出口所需的粘附分子，这对于完成裂解周期至关重要。该提案的目的是揭示TGDOC2.1在微元分泌中的作用和功能。这将提供令人兴奋的新见解，以了解不仅对弓形虫的发病机理，而且对所有其他Apicomplexan寄生虫的发病机理至关重要的机制，因为Ca2+依赖性的微神经分泌是整个门的保守的。同时，这将提供有关该途径潜力的见解由当前批准的药物针对）。在其他系统中，含有蛋白质的DOC2域域募集了膜融合机械（例如，SNARE和MUNC蛋白），以促进分泌囊泡与质膜的融合。但是，在TGDOC2.1中，没有与分泌机械相互作用的保守域是保守的。 TGDOC2.1的比较基因组学鉴定了跨APICOMPLEXA的四个保守序列块，突出了这些域的潜在至关重要的作用（块2包含DOC2域）。为了剖析tgdoc2.1的功能，我们将首先生成特定的抗血清或融合 - 重复蛋白的试剂，以在整个Ca2+依赖性的吞噬作用中建立其时空的亚细胞定位模式。这将表明哪种膜TGDOC2.1发挥其功能。此外，我们将建立有条件的TGDOC2.1敲低寄生虫线，可用于与TGDOC2.1结构域缺失突变体的功能互补研究。这将确定哪些域在膜易位和/或微原质分泌中起关键作用。在同一模型中，我们将测试Ca2+结合域中的点突变。 Ca2+ - 结合ASP残基将首先使用良好的异源哺乳动物组织培养模型映射。同时，要了解蛋白质TGDOC2.1在分泌过程中合作的蛋白质，我们将使用两种平行方法识别与TGDOC2.1相互作用的蛋白质。第一个是遗传酵母两种杂交系统，其中我们将使用tgdoc2.1作为诱饵来筛选tachyzoite cDNA库。在第二个中，我们将探索一种名为Bioid的新方法。在这种方法中，我们将将大肠杆菌Bira突变蛋白融合到TGDOC2.1并在寄生虫中表达。 BiRA突变体导致同一复合物中蛋白质的混杂生物赖以生物弥补，随后可以通过链霉亲和素富集和质谱法很容易识别。假定的TGDOC2.1通过两种方法确定的相互作用伙伴将通过寄生虫的共定位研究来验证。总体而言，我们将定义保守的微原质分泌过程背后的机制。