Elucidating Cellular Dependencies of O-GlcNAc Transferase Structure and Function

Abstract

O-GlcNAc transferase (OGT) is an essential enzyme and the most conserved glycosyltransferase in humans. Previous studies of OGT at both the cell and organismal level have modulated OGT with genetic deletion, pharmacological inhibition, or RNA interference. These studies utilized methods where phenotypes were scored following prolonged genetic perturbation or probes that exhibited off-target effects. Moreover, the authors attributed phenotypes associated with knockout or knockdown of OGT as being solely due to a lack of glycosyltransferase activity. However, OGT has numerous binding partners and has been observed in multiprotein complexes, suggesting noncatalytic roles that may regulate cellular physiology. In this thesis, I use next-generation chemical and genetic tools to investigate how changes in OGT’s catalytic and non-catalytic functions alter organellar and cellular physiology. First, I introduce this complex enzyme and previous attempts to define its role in cells. In Chapter 2, I collaborate with a graduate student to investigate how truncations of the tetratricopeptide repeat (TPR) domain of OGT alters its subcellular localization and subsequently cellular viability. In Chapter 3, I present a three-arm chemical genetic screen that separates OGT’s catalytic and non-catalytic synthetic lethal partners. In Chapter 4, I elucidate how a chemical probe that specifically inhibits OGT alters mitochondrial physiology on the scale of hours, suggesting OGT’s essentiality is linked to its role in mitochondrial homeostasis. Finally, in chapter 5 I conclude that OGT’s catalytic functions drive its essentiality and suggest future directions for OGT studies in cells.