Understanding the mechanism of adaptor protein engagement by OGT and its functional effects on glycosylation - 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 sites along the TPR domain and identify novel adaptor proteins for future mechanistic studies.
This information will enable the advancement of new strategies to selectively interrogate O-GlcNAc’s role on
specific substrates for future therapeutic applications.
