CAREER: A Genetic Method to Study Interactions Among Extracellular Proteins

Stanford University, Stanford CA

Investigators

Abstract

Extracellular glycoproteins mediate essential cellular recognition and binding events. This large and vital part of the proteome remains poorly understood due to challenges associated with studying glycosylated proteins. The objective of this CAREER project is to develop a method inspired by the yeast two-hybrid system, but optimized for use with post-translationally modified extracellular proteins. The new method is dubbed the ''Golgi two-hybrid assay.'' This strategy takes advantage of the fact that Golgi-resident glycosyltransferases can be subdivided into modular domains. In this new technique, protein-protein interactions will reconstitute the activity of a reporter glycosyltransferase. Technique implementation will begin with the demonstration that the reporter glycosyltransferase''s activity can be reconstituted by a known protein-protein interaction. These initial experiments will be followed by a calibration of the sensitivity and selectivity of the method. Finally, the method will be applied to the rapid identification of interaction partners for glycoproteins of interest. This new two-hybrid method is a key foundational tool to facilitate the study of glycosylation and glycoproteins. In addition, it will be adaptable for future needs such as secretome-wide interaction mapping, dissecting the contribution of specific glycan structures to known protein-protein interactions, directed molecular evolution to produce novel or improved interaction partners, and the discovery of small molecule inhibitors of adhesive interactions. Broader Impacts In addition to undergraduate student training and educational activities, the project will involve outreach to a local high school. By building a partnership with a local high school, minority high school students will be mentored and trained. Through summer laboratory employment, high school students will gain research experience and scientific confidence while they play integral roles in the development of modern technologies. Additionally, each summer, one local high school teacher will participate in laboratory research and have the opportunity to translate these technologies into classroom curriculum materials. These two types of outreach activities complement one another and are aimed at striking a balance between (1) making substantial commitments to individual students and (2) leveraging resources to reach as many students as possible. High school students, high school teachers, and undergraduate students will participate in all aims of this project, advancing the twin goals of (1) preparing students for future scientific careers and (2) ensuring the usability of the new two-hybrid method.