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

人类细胞表面蛋白和分泌配体之间相互作用的全局图

基本信息

批准号：
10478763
负责人：
Kenan Christopher GARCIA
金额：
$ 171.79万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-30 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10478763
关键词：
Address Affect Affinity Chromatography Avidity Award Binding Biochemical Biological Assay Biological Process Biomedical Research Cell Communication Cell Surface Proteins Cell physiology Cell surface Cells Cellular Assay Chimeric Proteins Code Color Communities Development Disease Enzyme-Linked Immunosorbent Assay Evaluation Extracellular Domain Extracellular Protein Funding Gene Expression Genes Goals High-Throughput DNA Sequencing Human Immune system Immunotherapy In Vitro Institution Knowledge Ligands Maps Mass Spectrum Analysis Mediating Methods Nervous system structure Neurons Orphan Peripheral Blood Mononuclear Cell Pharmaceutical Preparations Physiology Process Proteins Research Resources T-Lymphocyte Technology Testing Therapeutic Transmembrane Domain Yeasts anti-tumor immune response base cancer immunotherapy experimental study in vitro Assay in vivo innovation insight knock-down nanoparticle neoplastic cell nerve stem cell new technology new therapeutic target novel therapeutics organ growth programmed cell death ligand 1 programmed cell death protein 1 relating to nervous system screening single cell analysis single cell mRNA sequencing therapeutic target yeast two hybrid system

项目摘要

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.

项目概要/摘要该提案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，定义了人类细胞表面蛋白（CSP）胞外域之间的体外相互作用，由于细胞与细胞之间的相互作用，该图谱将对生物医学研究产生重大影响。由 CSP 介导的细胞因子是人类生理学的核心，控制着几乎所有受影响的生物过程 CSP 和分泌配体构成了大多数已确定的治疗靶点。近年来成功开发的互动伙伴知识对于评估 CSP 的治疗潜力，因为这种知识定义了它控制的生物过程。例如，PD-1 于 1992 年被确定为 T 细胞功能的负调节因子，但其作为 T 细胞功能靶点的价值癌症免疫疗法直到其配体 PD-L1 被鉴定并被发现是很久以后才变得清晰起来。我们不仅会生成人类之间体外相互作用的完整图谱。 CSP 和分泌蛋白，还评估这些相互作用在人体免疫细胞中的功能这是一个巨大的工程，因为人类大约有2000个单次跨膜。域 CSP 和 200 个“孤儿”分泌因子创建所有这些之间的成对相互作用图。蛋白质需要测试 480 万次相互作用，这超出了当前筛选方法的能力，因此。在学术机构执行此屏幕需要开发新技术。项目太大，传统 RO1 无法支持，但非常适合变革性研究在这里，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 和分泌的所有体外相互作用配体并在 5 年资助期内评估其中许多配体的功能。为此，我们首先将。使用颜色编码的珠子和高亲和力纳米颗粒对体外相互作用组筛选进行多重分析和敏化。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，其具有巨大的多重能力对于功能屏幕，多重单-。细胞命运扰动的细胞分析可以让我们评估许多不同配体对单个细胞的影响单个实验中的免疫系统和神经细胞此处描述的总体方法的基本原理。是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屏幕技术，然后使用最适合执行实际屏幕的技术。