Determining the Function of O-GlcNAc Protein Modification in Signal Transduction

University Of Minnesota-Twin Cities, Minneapolis MN

Investigators

Abstract

Intellectual Merit: Post-translational modification of cytosolic and nuclear proteins with O-linked ß-D-N-acetylglucosamine (O-GlcNAc) affects numerous processes in plants and animals. In plants, this modification is made by two O-linked N-acetylglucosamine transferases (OGTs), which in Arabidopsis thaliana are called SECRET AGENT (SEC) and SPINDLY (SPY). Knowledge about what processes are regulated and how this regulation occurs is limited and several lines of evidence suggest that, while O-GlcNAc function in plants and animals has similarities, there will be significant differences. For example, animals have a SEC-like OGT but lack the SPY-like OGT, which is unique to plants. Studies with sec and spy mutants indicate that O-GlcNAc modification affects numerous processes including response to hormones and light, embryo development, and regulation of flowering time but mechanistic details about how this regulation occurs are lacking. While the amount of O-GlcNAc modification to animal proteins increases and decreases in response to changes in hormonal and growth conditions, this has not been investigated in plants. Lack of information about which plant proteins are O-GlcNAc modified is a major impediment to understanding the role of this modification. The long-term goal of this project is to determine which plant processes are regulated by O-GlcNAc modification and elucidate the mechanisms of this regulation. Under the first aim of this proposal, the mass spectrometry-based methods will be optimized and used to identity O-GlcNAc modified proteins and the modification sites. The effects of growth conditions on the amount and distribution of the modification across the proteome will be determined. In addition, computational approaches will be used to identify signaling networks that are affected by O-GlcNAc modification. The final aim will test hypotheses about the networks where O-GlcNAc acts by determining if the functioning of these networks is affected in sec or spy mutants. Additional experiments will determine if SEC and/or SPY modifies selected O-GlcNAc modified proteins. Broader Impacts: This research will generate opportunities for high school, undergraduate and graduate students, and postdoctoral fellows to gain significant research experience and to develop their critical analysis, scientific problem solving and communication skills. High school students will be recruited from the Mentor Connection programs of local school districts. Undergraduates from a campus-wide Life Science Summer Undergraduate Research Program and students matriculating at the University of Minnesota will recruited to the lab. By mentoring high school students and undergraduates, laboratory members will gain teaching experience. To further engage undergraduates and the public, the principle investigator will give research talks at undergraduate institutions and participate in panel discussions and forums to educate the public about transgenic technology. He will co-instruct a BioArt course that involves projects using biological organisms to create art and also explores ethical considerations about working with living organisms. In addition, this research will contribute to scientific infrastructure by providing new methods for the identification of O-GlcNAc modified proteins.