A Global Map of Interactions Among Human Cell Surface Proteins and Secreted Ligands

项目概要/摘要 这项提议所面临的挑战是生成一个地图，即全球人类细胞表面相互作用组， 定义了人细胞表面蛋白（CSP）胞外结构域之间的体外相互作用， 分泌的蛋白质这张地图将对生物医学研究产生重大影响，因为细胞与细胞之间的相互作用 由CSP介导的是人类生理学的核心，控制着几乎所有受影响的生物过程。 疾病。CSP和分泌的配体构成了已经研究的大多数治疗靶标。 近年来成功开发。了解互动伙伴对于评估 CSP的治疗潜力，因为这种知识定义了它控制的生物过程。为 例如，PD-1在1992年被鉴定为T细胞功能的负调节剂，但其作为靶点的价值是 癌症免疫疗法直到很久以后才变得清晰，当它的配体PD-L1被鉴定并发现是 在肿瘤细胞上表达。我们不仅将生成一个完整的人类与哺乳动物之间体外相互作用的图谱， CSP和分泌的蛋白质，而且还评估了这些相互作用在人类免疫细胞中的功能。 和神经系统。这是一个巨大的工程，因为人类大约有2000个单跨膜 结构域CSP和200个“孤儿”分泌因子。创建一个所有这些之间的成对相互作用的地图 蛋白质需要测试480万个相互作用。这超出了当前筛选方法的能力，因此 在学术机构进行这种筛选将需要开发新技术。这 项目太大，无法由传统的RO 1支持，但非常适合变革性研究 奖励机制。在这里，我们提出了新的方法，既在体外生化筛选和体内多重 功能筛选，以便能够定义CSP和分泌的 配体，并在5年的资助期内评估其中许多的功能。为此，我们将首先 使用颜色编码的珠粒和高亲合力纳米颗粒进行多重和敏化体外相互作用组筛选。 然后，我们将开发将体外蛋白质相互作用筛选转化为高通量DNA的方法 测序屏幕，具有巨大的多路复用能力。对于功能屏幕，多路复用单- 细胞命运扰动的细胞分析可以使我们评估许多不同配体对单个细胞的影响。 免疫系统和神经细胞在一个单一的实验。此处所述总体方法的基本原理 它定义了一个逐步的过程，在这个过程中我们系统地开发和优化筛查技术，然后 使用最适合实际屏幕执行的技术。

PROJECT SUMMARY/ABSTRACT The challenge addressed by this proposal is to generate a map, the global human cell-surface interactome, that defines in vitro interactions among the extracellular domains of human cellsurface proteins (CSPs) and secreted proteins. This map will have a major impact on biomedical research, because cell-cell interactions mediated by CSPs are central to human physiology, controlling almost every biological process that is affected by disease. CSPs and secreted ligands comprise the majority of the therapeutic targets that have been successfully developed in recent years. Knowledge of interaction partners is essential for assessing the therapeutic potential of a CSP, since this knowledge defines the biological processes that it controls. For example, PD-1 was identified as a negative regulator of T cell function in 1992, but its value as a target for cancer immunotherapy only became clear much later, when its ligand PD-L1 was identified and found to be expressed on tumor cells. We will not only generate a complete map of in vitro interactions among human CSPs and secreted proteins, but also assess the functions of these interactions in cells of the human immune and nervous systems. This is a huge project, because there are about 2000 human single-transmembrane domain CSPs and 200 “orphan” secreted factors. Creation of a map of pairwise interactions among all of these proteins requires testing 4.8 million interactions. This is beyond the capacity of current screening methods, so execution of this screen at an academic institution will require the development of new technologies. This project is too large to be supported by a traditional RO1, but is perfectly suited to the transformative research award mechanism. Here we propose new ways to multiplex both in vitro biochemical screens and in vivo functional screens, so as to make it possible to define all in vitro interactions among CSPs and secreted ligands and to assess the functions of many of these within a 5-year funding period. To do this, we will first multiplex and sensitize in vitro interactome screens using color-coded beads and high-avidity nanoparticles. We will then develop methods to convert in vitro protein interaction screens into high-throughput DNA sequencing screens, which have a huge multiplexing capacity. For the functional screens, multiplexing single- cell analysis of cell fate perturbations can allow us to assess the effects of many different ligands on single immune system and neural cells in a single experiment. The rationale for the overall approach described here is that it defines a stepwise process in which we systematically develop and optimize screen technologies, then use the technology that performs best for execution of the actual screens.