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（由申请人提供）：尽管决定寄生虫成功或失败的分子机制为免疫提供了重要的见解，但迄今为止，很少有天然的宿主-寄生虫相互作用得到高度表征。我们已经开发了一种基于共同进化的宿主-寄生虫的模型系统。配对来分析果蝇免疫及其寄生黄蜂（Leptopilina spp.）的攻击武器库中的相互作用，该系统提供了复杂性和微妙性。解析先天免疫激活、沉默和颠覆，先天免疫功能从果蝇到人类都广泛保留，我们的长期目标是提供最独特的毒液。 Leptopilina spp. 的产物是免疫抑制微结构，称为细胞特异性药物递送和免疫治疗调节代表了我们对这些独特功能的研究的两种可能结果。 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，这项工作标志着我们首次对单一 VLP 蛋白质进行了功能研究。 Leptopilina 黄蜂在 VLP 蛋白质组中含量很高，并且具有假定的 GTP 结合和水解位点。在全基因组遗传相互作用筛选中进行分析时，这种 GTP 酶会导致与逆行受损相关的合成生长抑制。在目标 1 中，我们将确认这些遗传相互作用结果，然后在 GTP/GDP 锁定突变体以及对正常囊泡运输产生负面影响的突变的背景下测试它们。 -果蝇血细胞群体，以检查其亚细胞定位和蛋白质-蛋白质相互作用。我们将测试其对果蝇幼虫中 NF-κB 依赖性信号传导的影响。Toll-NF-κB 途径是激活的基础。果蝇先天免疫系统的细胞和体液臂，我们相信这种基于 GTP 酶的逆行运输调节可能会抑制正常的免疫信号传导。我们有关于配对模型系统方法的初步信息。通过强大的酵母筛选来研究细胞功能和靶向新型蛋白质，该方法提供了数千种遗传相互作用的信息，使我们能够在果蝇中设计适当且有效的实验计划。我们的目标是为 PI 提供深入的酵母技能。和果蝇遗传学、转基因、高通量全基因组遗传筛选、免疫组织化学和荧光成像将由跨学科环境中的该领域专家组成的团队支持，该团队将为 PI 提供高水平培训。 免疫、多模型系统、遗传学和先进的分子生物学技术。