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.
The goal of this project was to develop a new method to discover protein-protein interactions. Interactions between proteins underlie many biological processes; consequently, a variety of methods are available to discover novel protein binding partners for a protein of interest. However, the most robust and widely-used techniques are optimized for application to intracellular proteins, which lack key post-translational modifications including glycosylation and disulfide bond formation. We proposed to develop a complementary technique that would be designed for application to the secretome, the abundant class of secretory pathway and secreted proteins that are typically glycosylated and oxidized. Our method is analogous the traditional and widely-used yeast two-hybrid method for protein-protein interaction discovery, but instead of taking place in the nucleus of yeast, it takes place in the Golgi so we named it the Golgi two-hybrid method. Whereas the traditional two-hybrid method relies on the modularity and reassembly of transcription factor, our Golgi two-hybrid assay relies on the modularity and reassembly of a Golgi-resident glycosyltransferase, Och1. We developed three different moderate-to-high throughput assays to measure and select for yeast with high levels of Och1 activity. We also showed that the modular Och1 could be separated into two polypeptides, which were then fused to potential interaction partners, which we term bait and prey. Interaction between bait and prey reassembled active Och1, which could be detected by any of our assays. In published work, we showed that we could use the Golgi two-hybrid method to detect known protein-protein interactions. Although we have not yet successfully apply this method to secretome proteins, as intended, we made the surprising and fortuitous discovery that it could be used to detect low-affinity interactions between transcription factors, another class of proteins that cannot be studied by the traditional two-hybrid method. Thus, the most significant intellectual merit outcome is the discovery that the new Golgi two-hybrid method can fill an important gap in protein-protein interaction discovery. During the remainder of the granting period, we focused our work in two areas: (1) collaborative projects where we use the Golgi two-hybrid assay to to define binding partners for proteins of interest, and (2) improved methods to introduce cDNA libraries into our Golgi two-hybrid system for de novo interaction discovery. Work in both of these areas is ongoing. This project also had a variety of broader impacts in the areas of education and scientific training. The Golgi two-hybrid project provided a training environment for the postdoctoral fellows and graduate students working in this research group. In addition, we hosted several minority summer undergraduate students (through the SURF program), who have now gone on to graduate school in highly competitive programs, including Harvard and UT Southwestern. Working with Collin County Community College (now Collin College), we hosted a co-op student from the Biotechnology Associate’s Degree program. The PI has participated and continues to participate in educational outreach, including on-campus presentations to visiting high school students through the STARS program and development of a science outreach program for kindergarteners in a local elementary school with a "minority-majority" (78% Hispanic; 9% black) population.
