Understanding tetratricopeptide repeats of O-GlcNAc transferase

Abstract

The modification of serine and threonine residues of intracellular proteins with O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) was serendipitously discovered in 1984. During the following decades it became clear that intracellular O-GlcNAc levels respond to various stimuli, suggesting that this modification is dynamic and may represent a novel cellular signalling mechanism. Despite the multitude of proteins now known to be modified by O-GlcNAc, only a single pair of enzymes modulate this modification: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Intriguingly, OGT has also recently been shown to have a secondary function, catalysing the proteolysis of the transcriptional regulator protein host cell factor-1 (HCF-1). OGT contains an N-terminal tetratricopeptide repeat (TPR) domain, with some evidence to suggest that this domain plays a role in substrate and adaptor protein binding. However, our understanding of the mechanisms underlying OGT substrate recognition and specificity remains limited. The work described in this thesis was carried out with the aim of understanding how the O-GlcNAc cycling enzymes achieve substrate recognition and specificity, with a particular focus on OGT and its TPR domain.

In the third chapter of this thesis, I describe the preliminary characterisation of the Gram-positive bacterium Thermobaculum terrenum as a potential reductionist model for the O-GlcNAcylation system where OGT-substrate interactions can be studied. Structural characterisation of T. terrenum OGT suggested that this enzyme is a catalytically competent OGT orthologue, while treatment of T. terrenum with OGT inhibitors demonstrated that this enzyme is required for bacterial growth. Similarly, using enzyme assays and structural characterisation of T. terrenum OGA I show that this enzyme is a bona fide O-GlcNAcase. In chapter four, the role of the OGT TPR domain was further investigated through biochemical characterisation of two novel human intellectual disability (ID)-associated OGT mutations that map to the TPR domain: OGTR284P and OGTΔ155-177. Investigations of patient-derived fibroblasts revealed that although OGT levels are reduced, downregulation of OGA expression appears to maintain O-GlcNAc homeostasis. Additionally, in vitro activity assays performed on OGTR284P demonstrated that this mutation resulted in non-specific reduction of both the glycosyltransferase and HCF-1 proteolytic maturation activities of OGT. Lastly, in chapter five, structural characterisation of a previously characterised ID-associated OGT TPR domain mutation, OGTL254F, is presented. Detailed comparative crystallographic analysis of the wild type and mutant TPR domain showed that the L254F mutation causes a subtle local structural distortion at the mutation site that propagates through the entire TPR domain, resulting in a substantial displacement of the most N-terminal portion of the TPR superhelix. In addition, molecular dynamics simulations of this mutant displayed markedly increased structural plasticity compared with the wild type. However, the impact of this mutation on the dual function of OGT remains unclear.

Overall, this work provides initial progress towards understanding the effect of ID-associated mutations on OGT and its TPRs in particular, and suggests that analysis of the impact of these mutations on interactions between OGT and its substrates and/or interacting partners may prove to be a fruitful avenue of research. Furthermore, this work has established a useful reductionist model to further investigate not only the questions of how the OGT TPRs mediate protein-protein interactions and thereby mediate OGT substrate specificity, but also to probe the broader role of the O-GlcNAc system at the organism level.

Date of Award	2017
Original language	English
Awarding Institution	University of Dundee
Supervisor	Daan van Aalten (Supervisor)