Monday, May 6, 2024
5/6/2024
Project Summary/Abstract Cell-cell interactions mediated by cell-surface proteins (CSPs) are central to human physiology, controlling assembly and maintenance of organs and tissues. The human genome has about 3700 genes encoding CSPs. In collaboration with a group at Stanford, one of the P.I.s conducted in vitro extracellular “interactome” screens that defined binding partners for 550 human CSPs, and he is currently working on a global in vitro screen. Such screens, which use soluble extracellular domain (ECD) fusion proteins, are most useful for single- transmembrane (TM) CSPs. Many CSPs with multiple TM domains, such as G protein-coupled receptors, have ECDs composed of discontinuous loops, making it difficult to express them in a soluble form. There are about 1700 genes encoding such CSPs. Identification of binding partners for multi-span CSPs will require the use of screens in which intact proteins are expressed on cellular or vesicular membranes. We plan to develop technology for a “library-on-library” cell-based screen for CSP interactions that can identify many binding partners in a single experiment. We hope to use this method to define interactions for CSPs expressed in the human nervous system. Our proposed method uses a new technology, developed by the other P.I., for making engineered extracellular vesicles, called eVLPs, that display single CSPs at high density on their surfaces. We plan to make a pool of eVLPs, each of which encapsulates a unique RNA barcode. Each eVLP will display one member of a library of target CSPs. In parallel, we will generate a cell library in which each cell expresses one CSP from the library. The eVLP pool will be mixed with the cell library, and we will select cells that have fused with an eVLP whose displayed CSP binds to the CSP on the cell surface. Fusion after binding is catalyzed by a mutant VSVG protein, and leads to expression of a fluorescent marker. We will then analyze the sorted cells by single-cell RNA sequencing. By recovering the barcode sequence, which identifies the CSP on the donor eVLP, and the sequence of the CSP expressed by the recipient cell, we will identify candidate CSP interactions that can later be validated using other methods. We plan to attain these objectives of this application through the following Specific Aims. Aim 1: Efficiently incorporate barcode RNAs into eVLPs. Aim 2: Use eVLPs to conduct pilot screens to identify known ligand-receptor pairs. We will determine whether interactions between a CSP on an eVLP and a CSP on recipient cells can lead to membrane fusion and allow sorting and sequencing of cells transduced by the eVLP. We will conduct a small pilot screen to determine if the cell-based screen can identify interactions that we previously found through in vitro screens. The expected outcome of the proposed research will be the development of a new method for library-on-library cell-based interactome screening. This will have a significant positive impact, in that it will allow our group and others to identify many new ligand-receptor interactions in the nervous system and elsewhere. Some of these interactions might define new therapeutic targets.
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