PIPs: a new means to identify effector-target interactions

PIP：识别效应器-目标相互作用的新方法

• 批准号: 8777643
• 负责人: CAMMIE LESSER
• 金额: $ 21.75万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-11 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8777643
• 关键词: Address; Bacteria; Binding; Biochemical; Biological Assay; Cell physiology; Cells; Co-Immunoprecipitations; Communities; Coupled; Cytosol; Data; Detection; Development; Disease; Exhibits; Generations; Genetic; Goals; Gold; Growth; Human; Immune; Individual; Infection; Life; Mammalian Cell; Mediating; Membrane; Microscopy; Minority; Molecular; Molecular Chaperones; Pathogenesis; Phenotype; Proteins; Recruitment Activity; Regulation; Reovirus; Role; Scaffolding Protein; Specificity; System; Testing; Therapeutic Agents; Virulence; Yeasts; base; enteric pathogen; follow-up; genetic selection; high throughput screening; innovation; insight; loss of function; microbial; novel; novel therapeutics; pathogen; protein protein interaction; public health relevance; yeast protein; yeast two hybrid system

DESCRIPTION (provided by applicant): Microbial pathogens often co-opt host cellular processes to promote their own survival, replication and spread. For example, many Gram-negative bacterial pathogens utilize specialized type 3 secretion systems to directly inject tens of proteins, referred to as effectors, directly into host cells. Studies devoted to determining the roles of these proteins in pathogenesis as well as their molecular modes of action have been instrumental in advancing our understanding of the means by which pathogens establish an infection and cause disease. Nevertheless, a function for only a minority of effectors is current known, as it is often challenging to determine their roles in pathogenesis. Effectors can be difficult to study given that their sequence uniqueness in general provides no clues as to their function and loss of expression of individual effectors often does not result in a detectable phenotype, as they often act in a functional redundant manner. Furthermore, genetic approaches to identify roles of effectors are similarly complicated given that the bacteria deliver tens to hundreds of proteins into host cells. For these reasons, to gain insights regarding the roles of effectors in pathogenesis, many groups initially focus their efforts on biochemical approaches aimed at identifying mammalian proteins that directly bind the effectors, an approach that also has it's caveats. For example, the relatively low-levels of effectors injected into host cells coupled with their tendency to act catalytic thus presumably only transiently with target proteins complicates co-immunoprecipitation based approaches, particularly in the context of an infection. Similarly, even targeted assays directed at following up candidate interacting proteins can be difficult, as the heterologous expression of effectors in yeast and mammalian cells is often toxic. We recently developed the Protein Interaction Platform assay or PIP, a powerful and innovative means to study binary protein interactions. PIP consistently outperforms Y2H in detecting interactions between effectors and their interacting proteins. Our long-term goal is to develop a large comprehensive set of yeast strains, each of which express a mammalian protein fused to ¿NS, that can be used in small or large-scale studies to identify new effector target proteins. However, such an endeavor is a huge undertaking and requires additional extensive supportive preliminary data. Here, towards this goal, we propose to compare the ability of PIP and Y2H to detect additional known effector-target interactions (Aim 1), conduct a small-scale screen to identify new candidate interactions between effectors and mammalian proteins involved in innate immune regulation (Aim 2) and convert PIP to a genetic selection (Aim 3).

描述（由适用提供）：微生物病原体通常会选择宿主细胞过程，以促进其自身的生存，复制和扩散。例如，许多革兰氏阴性细菌病原体利用专门的3型分泌系统直接注入了数十种蛋白质，称为作用，直接将其直接注入宿主细胞中。致力于确定的研究 这些蛋白质在发病机理及其分子作用模式中的作用对我们对病原体建立感染并引起疾病的手段的理解发挥了作用。然而，目前只有少数效应的功能，因为通常要确定其在发病机理中的作用。效应子可能很难研究其序列唯一性一般没有提供有关其功能的线索和单个效应表达的丧失，通常不会导致可检测的表型，因为它们通常以功能多余的方式起作用。此外，鉴于细菌递送的遗传方法鉴定作用的作用是同样复杂的 数十至数百种蛋白质进入宿主细胞。由于这些原因，为了了解效应在发病机理中的作用的见解，许多群体最初将精力集中在旨在识别直接结合效果的哺乳动物蛋白质的生化方法上，这种方法也具有警告。例如，注射到宿主细胞中的相对低水平的效应以及其作用催化作用的趋势，大概仅与靶蛋白瞬时相关，使基于共免疫沉淀的方法复杂化，尤其是在感染的背景下。同样，即使是旨在跟踪候选蛋白质相互作用蛋白质的有针对性测定也可能很困难，因为酵母和哺乳动物细胞中作用的异源表达通常是有毒的。我们最近开发了蛋白质相互作用平台分析或PIP，这是研究二元蛋白质相互作用的强大而创新的手段。 PIP在检测效应及其相互作用蛋白之间的相互作用方面始终优于Y2H。我们的长期目标是开发一大批综合的酵母菌菌株，每种酵母菌菌株表达与ns融合的哺乳动物蛋白质，可在小型或大型研究中使用，以鉴定新的效应靶蛋白。但是，这样的努力是一项艰巨的任务，需要更多的广泛支持性初步数据。在此，为了实现这一目标，我们建议比较PIP和Y2H检测其他已知效应靶标相互作用（AIM 1）的能力（AIM 1），进行一个小规模的屏幕，以识别与先天免疫调节有关的效应与哺乳动物蛋白之间的新候选蛋白相互作用（AIM 2）并转化PIP转化为遗传选择（AIM 3）。