Modulation of Retrograde Transport by a Novel Parasite-Derived GTPase

新型寄生虫衍生的 GTP 酶对逆行转运的调节

• 批准号: 8987333
• 负责人: Mary Ellen Heavner
• 金额: $ 2.71万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-16 至 2018-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8987333
• 关键词: Ablation; Acute; Affect; Biogenesis; Bioinformatics; Biological Assay; Biological Models; Blood Cells; Cell physiology; Cells; Complex; Computer Analysis; Custom; Databases; Drosophila genus; Drosophila melanogaster; Drug Delivery Systems; Eating; Educational Status; Failure; Figs - dietary; Fruit; GTP Binding; Gene Expression Profile; General Practitioners; Genetic; Genetic Screening; Genetic screening method; Gland; Goals; Growth; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Homology Modeling; Host Defense; Human; Hydrolysis; Immune; Immune system; Immunity; Immunohistochemistry; Immunosuppressive Agents; Infection; Inflammatory; Insecta; Investigation; Larva; Microscopic; Molecular; Molecular Biology Techniques; Mutation; Nature; Nucleic Acids; Organism; Outcome; Parasites; Pathway interactions; Population; Process; Proteins; Proteome; Proteomics; Race; Reaction; Relative (related person); Repression; Signal Transduction; Site; Subgroup; System; Testing; Therapeutic; Transgenic Organisms; Venoms; Vesicle; Virulence; Virulence Factors; Virulent; Virus; Virus-like particle; Wasp Venoms; Wasps; Work; Yeasts; arm; base; cellular targeting; collaborative environment; comparative; design; egg; fighting; flexibility; fluorescence imaging; fly; genome-wide; immune activation; immunoregulation; in vivo; innate immune function; insight; killings; mutant; novel; particle; protein aminoacid sequence; protein protein interaction; protein transport; retrograde transport; skills; success; tool; trafficking; translational medicine

DESCRIPTION (provided by applicant): Few natural host-parasite interactions have been highly characterized to date, even though the molecular mechanisms that determine parasite success or failure offer critical insights into immunity. We have developed a model system based on a coevolved host-parasite pair to analyze the reciprocal interactions underlying immunity in Drosophila melanogaster and the attack arsenals of its parasitic wasps (Leptopilina spp.). This system offers the complexity and subtlety to dissect innate immune activation, silencing, and subversion. D. melanogaster genetics are tractable, fast, and low-impact. Innate immune functions are widely conserved from fly to human and our long-term goal is to provide translational results. The most unique venom products of Leptopilina spp. are immunosuppressive microstructures, referred to as VLPs. Cell-specific drug delivery and therapeutic immune modulation represent two possible outcomes our investigations of the function of these unique parasite-derived particles. The protein composition of VLPs is central to the attack success of these parasites of Drosophila. This work marks the first functional investigation into a singular VLP protein. We have selected a gene product that is expressed only by the most virulent of Leptopilina wasps, is highly abundant in the VLP proteome, and possesses a putative site for GTP binding and hydrolysis. When analyzed in a genome-wide genetic interaction screen, this GTPase causes synthetic growth repression associated with impaired retrograde transport. In Aim 1, we will confirm these genetic interaction results and then test them in the context of GTP/GDP-locked mutants, as well as mutations that negatively impact normal vesicular trafficking. In Aim 2, we will express this protein in sub-populations of Drosophila blood cells to examine its subcellular localization and protein-protein interactions. We will test its impact on NF-κB-dependent signaling in fly larvae. The Toll- NF-κB pathway underlies activation of both the cellular and humoral arms of Drosophila's innate immune system and we believe that this GTPase-based modulation of retrograde transport may suppress normal immune signaling. The strategies utilized in this work depend on a paired model system approach. We have obtained preliminary information on the cellular function and targeting of a novel protein via a powerful yeast screen that provides information on thousands of genetic interactions. This approach has allowed us to design appropriate and efficient experimental plans in Drosophila. Our aims will provide the PI with in-depth skills in yeast and Drosophila genetics, transgenics, high-throughput genome-wide genetic screens, immunohistochemistry, and fluorescence imaging. This work will be strongly supported by a team that includes experts in the field in an interdisciplinary environment that will provide high-level training to the PI in immunity, multiple model system, genetics, and advanced molecular biology techniques.

描述（由适用提供）：迄今为止，很少有天然的宿主 - 寄生虫相互作用被高度表征，即使决定寄生虫成功或失败的分子机制为免疫学提供了关键的见解。我们已经开发了一个基于共同发展的宿主寄生虫对的模型系统，以分析果蝇中果蝇中免疫学的相互作用相互作用，以及其寄生黄蜂的攻击砷（Leptopilina spp。）。该系统提供了复杂性和微妙之处，以剖析先天免疫激活，沉默和颠覆。 D. melanogaster遗传学是可探讨，快速和低影响的。天生的免疫功能从苍蝇到人都得到了广泛保守，我们的长期目标是提供翻译结果。 Leptopilina spp最独特的毒液产品。是免疫抑制的微观结构，称为VLP。细胞特异性的药物输送和治疗性免疫调节代表了我们对这些独特的寄生虫衍生颗粒功能的投资。 VLP的蛋白质组成对于果蝇这些寄生虫的攻击成功至关重要。这项工作标志着对单数VLP蛋白的首次功能投资。我们选择了仅由最有毒的瘦素黄蜂表达的基因产物，在VLP蛋白中高度丰富，并具有用于GTP结合和水解的假定位点。当在全基因组遗传相互作用筛选中进行分析时，该GTPase会导致与逆行转运受损相关的合成生长表达。在AIM 1中，我们将确认这些遗传相互作用结果，然后在GTP/GDP锁定的突变体的背景下对其进行测试，以及对正常囊泡运输的负面影响。在AIM 2中，我们将在果蝇血细胞的亚种群中表达这种蛋白质，以检查其亚细胞定位和蛋白质 - 蛋白质相互作用。我们将测试其对蝇幼虫中NF-κB依赖性信号传导的影响。 TOLL-NF-κB途径是果蝇先天免疫点系统的细胞和体液臂激活的基础，我们相信这种基于GTPa​​se的逆行转运调节可以抑制正常的免疫抑制信号传导。这项工作中使用的策略取决于配对的模型系统方法。我们通过强大的酵母筛选获得了有关细胞功能和靶向新蛋白的初步信息，该筛网提供了数千种遗传相互作用的信息。这种方法使我们能够在果蝇中设计适当有效的实验计划。我们的目标将为PI提供酵母和果蝇遗传学，转化学，高通量全基因组遗传筛查，免疫组织化学和荧光成像的深入技能。这项工作将得到一个团队的强烈支持，该团队在跨学科环境中包括该领域的专家，该团队将为PI提供高级培训 免疫，多个模型系统，遗传学和晚期分子生物学技术。