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

项目摘要/摘要 这一提议所解决的挑战是生成一张地图，即全球人类细胞表面相互作用组， 它定义了人细胞表面蛋白(CSPs)的胞外域和 分泌的蛋白质。这张图将对生物医学研究产生重大影响，因为细胞之间的相互作用 CSP是人类生理的中心，几乎控制着每一个受影响的生物过程 被疾病折磨。CSPs和分泌配体构成了大多数已被治疗的靶点 近几年发展成功。互动合作伙伴的知识对于评估 CSP的治疗潜力，因为这一知识定义了它所控制的生物过程。为 例如，PD-1在1992年被确定为T细胞功能的负调节因子，但其作为靶点的价值 癌症免疫治疗直到很久以后才变得清晰，当时它的配体PD-L1被发现是 在肿瘤细胞上表达。我们不仅将生成人类之间体外相互作用的完整地图 CSPs和分泌蛋白，还评估了这些相互作用在细胞中的功能对人类免疫的影响 和神经系统。这是一个巨大的项目，因为大约有2000个人类单细胞跨膜 域CSP和200个“孤儿”分泌因子。创建所有这些元素之间成对交互的地图 蛋白质需要测试480万种相互作用。这超出了当前筛查方法的能力，因此 在学术机构中执行这一屏幕将需要开发新技术。这 项目太大，传统的RO1无法支持，但非常适合变革性研究 奖励机制。在这里，我们提出了体外生化筛选和体内多重筛选的新方法。 功能筛选，以便能够定义CSP和分泌物之间的所有体外相互作用 在5年的资助期内，评估其中许多配体的功能。要做到这一点，我们首先 使用颜色编码的珠子和高亲和力的纳米颗粒对体外交互作用组屏幕进行多路传输和增敏。 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让我们评估许多不同的配体对单个 免疫系统和神经细胞在一次实验中。这里描述的总体方法的基本原理 它定义了一个循序渐进的过程，在这个过程中，我们系统地开发和优化屏幕技术，然后 使用执行实际屏幕性能最好的技术。

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.