The modification of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is found on many proteins in the nucleus and cytoplasm. O-GlcNAc has been implicated in many physiological processes but much remains to be learned about the effects of this modification on protein function. In this thesis I detail two studies aimed to improve our understanding of protein modification by O-GlcNAc. First, I describe a bioinformatics study focused on uncovering the protein structural features that favour O-GlcNAcylation by the enzyme O-GlcNAc transferase. The search for a sequence or structural motif to be found amongst the many substrates O-GlcNAcylated by OGT is a path well-trodden. On the basis of our analysis of site mapping data accumulated from the literature and also through targeted site mapping of an entirely intrinsically disordered protein by mass spectrometry, I counter argue that OGT preferentially targets substrates which can be intrinsically disordered.Second, I describe a study aimed to gain insight into cellular proteomic response due to lowering of O-GlcNAc levels. There is mounting evidence O-GlcNAcylation is both linked with protein folding and intracellular proteome stability. While stability is measured by turnover it is also tied to misfolding. We investigated a possible relationship between lowered O-GlcNAc levels and instability of a segment of the proteome using stable isotope labelling by amino acids in cell culture mass spectrometry.
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Thesis advisor: Vocadlo, David
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