Biochemical Characterization of Interactions Between O-GlcNAc Transferase and Its Protein Binding Partners

Abstract

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that is solely responsible for the post-translational modification of O-GlcNAcylation, the addition of N-acetyl-D-glucosamine (GlcNAc) onto serine and threonine sidechains on thousands of proteins in the nucleus and cytoplasm. OGT catalyzes another post-translational modification: the cleavage of host cell factor 1 (HCF-1), which it does via O-GlcNAcylation of a glutamate residue in one of six highly conserved proteolytic repeats. OGT also engages in a wide variety of protein complexes, facilitated primarily by its superhelical tetratricopeptide repeat (TPR) domain. Previous work from the lab has demonstrated that OGT’s O-GlcNAcylation and noncatalytic scaffolding functions are essential for cellular proliferation. In this thesis, I build on previous notions of interactions between OGT and both its protein substrates and non-substrate interactors, defining features that drive these essential functions. Chapter 2 details a series of work performed in collaboration with other members of the Walker lab, studying the effects of mutations and truncations within the TPR domain on OGT’s functions and its ability to support cell proliferation; we show that truncation of OGT’s N-terminal TPRs impacts viability and all three functions, while preventing OGT homodimerization leads to increased cell growth. Additionally, in that chapter, I show that changes to protein-interacting features in the TPR lumen do not impact all of OGT’s substrates equally. In Chapter 3, I harness TR-FRET technology to develop a quantitative binding assay for OGT-substrate interactions. Using that assay, I identify a polypeptide derived from HCF-1’s proteolytic repeats, HCF3R, that has picomolar affinity and inhibits OGT in vitro and in cells. I then modify this probe to create a cellular OGT inhibitor that can be switched on and off by addition of two small molecules. In Chapter 4, I use a combination of biochemical and structural tools to interrogate the nature of the OGT-HCF3R interaction, finding that the medial TPR region (TPRs 6-8) contains features that are essential for the observed phenotypes. Taken together, this work demonstrates methods that can be used to further probe OGT’s functions, and insights by which new understanding of OGT’s protein-protein interactions can be derived.