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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：
10710033
负责人：
Kenan Christopher GARCIA
金额：
$ 270.14万
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/10710033
关键词：
Acceleration 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 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 Neurons Orphan Peripheral Blood Mononuclear Cell Pharmaceutical Preparations Physiology Process Protein Secretion Proteins Research Resources T-Lymphocyte Technology Testing Therapeutic Transmembrane Domain Yeasts anti-tumor immune response cancer immunotherapy experimental study in vitro Assay in vivo innovation insight knock-down nanoparticle neoplastic cell nerve stem cell neural new technology new therapeutic target novel therapeutics organ growth programmed cell death ligand 1 programmed cell death protein 1 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，以及分泌的蛋白质。这张图将对生物医学研究产生重大影响，因为细胞与细胞的相互作用由 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通过疾病。 CSP 和分泌配体构成了已发现的大多数治疗靶点近年来开发成功。了解互动伙伴对于评估互动伙伴至关重要 CSP 的治疗潜力，因为这种知识定义了它控制的生物过程。为了例如，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。表达于肿瘤细胞上。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和神经系统。这是一个浩大的工程，因为人类的单次跨膜约有2000个域 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。这超出了现有筛选方法的能力，因此在学术机构执行此屏幕需要开发新技术。这项目太大，传统 RO1 无法支持，但非常适合变革性研究奖励机制。在这里，我们提出了多重体外生化筛选和体内生化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，我们首先要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，具有巨大的多重能力。对于功能屏幕，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配体对单个细胞的影响免疫系统和神经细胞在一次实验中。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。