PIPs: a new means to identify effector-target interactions

描述(由申请人提供)：微生物病原体通常选择宿主细胞过程来促进自身的生存、复制和传播。例如，许多革兰氏阴性细菌病原体利用专门的3型分泌系统将数十种蛋白质直接注入宿主细胞，称为效应器。致力于确定 这些蛋白在发病机制中的作用以及它们的分子作用模式有助于促进我们对病原体建立感染和导致疾病的方式的理解。然而，目前只有少数效应器的功能是已知的，因为确定它们在发病机制中的作用往往是具有挑战性的。效应器可能很难研究，因为它们的序列唯一性通常不提供关于它们的功能的线索，并且单个效应器的表达的丢失通常不会导致可检测的表型，因为它们经常以功能冗余的方式起作用。此外，识别效应器角色的遗传方法也同样复杂，因为细菌提供 几十到几百种蛋白质进入宿主细胞。出于这些原因，为了深入了解效应器在发病机制中的作用，许多小组最初将工作重点放在旨在识别直接结合效应器的哺乳动物蛋白质的生化方法上，这种方法也有其局限性。例如，注射到宿主细胞中的效应物水平相对较低，再加上它们具有催化作用的倾向，因此推测只与目标蛋白短暂地结合使基于免疫共沉淀的方法复杂化，特别是在感染的情况下。同样，由于效应物在酵母和哺乳动物细胞中的异源表达通常是有毒的，即使是针对跟踪候选相互作用蛋白的靶向分析也可能是困难的。我们最近开发了蛋白质相互作用平台分析或PIP，这是一种研究二元蛋白质相互作用的强大而创新的手段。在检测效应器及其相互作用的蛋白质之间的相互作用方面，PIP始终优于Y2H。我们的长期目标是开发一大套全面的酵母菌株，每一种酵母菌株都能表达一种与�NS融合的哺乳动物蛋白，可以用于小规模或大规模的研究，以确定新的效应靶蛋白。然而，这样的努力是一项巨大的事业，需要更多广泛的支持性初步数据。在这里，为了这个目标，我们建议比较PIP和Y2H检测其他已知效应器-靶相互作用的能力(目标1)，进行小规模筛选以确定效应器和参与先天性免疫调节的哺乳动物蛋白之间新的候选相互作用(目标2)，并将PIP转换为基因选择(目标3)。

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).