PROJECT SUMMARY/ABSTRACT The identification of O-linked beta-N-acetylglucosamine (O-GlcNAc) modified proteins in the nucleus and cytoplasm overturned the paradigm that glycosylated proteins are only found in the secretory pathway of eukaryotes. Since then, O-GlcNAc modifications, installed by the O-GlcNAc transferase (OGT) enzyme, have been identified on proteins involved in almost all cellular processes. O-GlcNAc levels rise upon increase of glucose levels, and perturbations in protein O-GlcNAcylation has been implicated in diseases caused by protein misregulation, such as cancer and Alzheimer’s disease. It has been speculated that methods to regulate O- GlcNAcylation levels on targeted substrates would be therapeutically advantageous. To date, over one thousand protein targets have been identified, however the mechanisms by which OGT chooses those substrates eludes scientists, making it challenging to develop effective therapeutic interventions. Substrate selection does not occur at the active site of OGT. Instead, OGT’s N-terminal tetratricopeptide repeat (TPR) domain has been implicated in substrate selection through two proposed mechanisms, either through 1) intrinsic interactions with substrates and/or 2) interactions with substrates mediated by adaptor protein binding that alter OGT’s enzymatic activity. The TPR domain contains 13.5 repeats that form a unique superhelix with two 100 Å long binding surfaces, the concave, lumenal surface that has been implicated in direct substrate binding and a convex, solvent-exposed surface that we hypothesize engages non-substrate protein interactors, such as adaptors. While several studies have provided insights into intrinsic substrate binding, adaptor-mediated substrate selection mechanisms are poorly understood due to the limited tools for selectively capturing non-substrate interactions. I propose experiments to identify unique adaptor binding sites along the solvent-exposed surface of OGT’s TPR domain and to develop strategies to interrogate the role of adaptor interactions in OGT substrate selection. In Aim 1, we will use a library of photoactivatable unnatural amino acid (UAA)-containing OGT constructs to covalently capture known adaptor proteins and generate a map of adaptor binding sites along the solvent-exposed surface of the TPR domain. Additionally, we will use TPR mutants and glycotransferase assays to interrogate the functional consequences of disrupting the OGT-adaptor binding interfaces on the glycosylation of individual substrates. In Aim 2, we will use the same library of UAA-containing OGT constructs to covalently capture novel TPR-surface interactors from whole cell extracts and develop a two-step screening strategy to separate adaptor proteins that alter OGT’s activity towards protein substrates from scaffolding proteins that do not alter OGT’s substrate glycosylation activity upon binding. Results from this study will provide the first comprehensive map of non- substrate binding s...