Engineering a proximity-directed O-GlcNAc transferase to control O-GlcNAc signaling

Abstract

O-GlcNAc is a ubiquitous monosaccharide posttranslational modification analogous to phosphorylation found on thousands of nuclear and cytoplasmic proteins involved in every major cellular process. Dysregulation of O-GlcNAc signaling contributes to the development and progression of several chronic diseases, though the exact molecular mechanisms driving these effects have remained elusive. A major bottleneck towards progress in the O-GlcNAc field is the absence of a facile method to increase O-GlcNAc on select proteins to determine the functional outcomes of O-GlcNAc modification. In this dissertation, I describe my efforts to generate a new protein tool that employs nanobodies and protein engineering of the O-GlcNAc Transferase (OGT) to control O-GlcNAc signaling in live cells. I begin in chapter 1 by providing a historical overview of the key concepts driving the O-GlcNAc field from before its inception to the present day. This is followed by the major concepts, applications, and current developments in the single-chain antibody field in Chapter 2. In Chapter 3, I detail the development of a nanobody-OGT fusion to target tagged and endogenous proteins to increase their levels of O-GlcNAc through a proximity-directed mechanism. Application of the nanobody-OGT technology to the methylcytosine dioxygenase TET3 reveals a scaffolding function for OGT in the regulation of TET3 localization. To further improve the nanobody-OGT system, advances in nanobody engineering are described to gain spatiotemporal control of O-GlcNAc signaling. In the fourth chapter, I study several structural features of OGT. I describe the effects of truncations of the tetratricopeptide (TPR) domain of OGT in substrate and glycosite selection in cells. I additionally explore mutations in the TPR domain, catalytic site, and intervening domain of OGT and their effects on activity, localization and selection of substrates and glycosites for future protein engineering of OGT. I conclude with a discussion of emerging concepts in the O-GlcNAc field and how the nanobody-OGT technology and single-chain antibodies could be harnessed to gain additional insight into this ubiquitous and important posttranslational modification for the advancement of basic discoveries in the O-GlcNAc field.