Functional and Mechanistic Insights into O-Glycosyl Modification of the Plant Transcription Factor SPATULA

Curran-French, Seamus (2024) Functional and Mechanistic Insights into O-Glycosyl Modification of the Plant Transcription Factor SPATULA. Doctoral thesis, University of East Anglia.

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Abstract

Modification of nucleocytoplasmic proteins by O-Linked-β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous and essential process in all eukaryotes. O-GlcNAcylation serves as a vital nutrient/stress sensor to regulate numerous fundamental processes including transcription, cell signalling, translation, protein trafficking, and cell division. In animals, cycling of O-GlcNAc is controlled by just two enzymes: O-GlcNAc transferase (OGT) which catalyses the addition of O-GlcNAc, and O-GlcNAcase (OGA) which removes the moiety from targets. Arabidopsis thaliana has two OGT homologs - SECRET AGENT (SEC) and SPINDLY (SPY). While SEC is a true OGT enzyme catalysing the addition of O-GlcNAc, SPY was recently identified as a novel type of O-fucose transferase (POFUT). Despite the importance of O-glycosyl-based modifications little is known about how OGT enzymes select such a diverse set of targets with seemingly no sequence or structural homology. This work investigated how SEC and SPY can regulate the activity of the basic Helix-Loop-Helix (bHLH) transcription factor SPATULA (SPT) – a key regulator of cell growth dynamics in plants - demonstrating that these modifications work synergistically to enhance the affinity of SPT for the kinase PINOID (PID) gene locus. This work also provides evidence that a region within the tetratricopeptide repeat (TPR) domain of SEC and SPY interacts with SPT which may confer substrate selectivity. Extensive biochemical characterisation of SPT and SPY was also carried out and a recombinant SPT-SPY complex formed in vitro, with preliminary cryogenic electron microscopy (cryo-EM) data suggesting a high-resolution structure of SPT bound to SPY may be achieved to elucidate the biochemical mechanism of protein substrate recognition by SPY and OGTs more generally.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Kitty Laine
Date Deposited:	03 Jun 2025 12:26
Last Modified:	03 Jun 2025 12:26
URI:	https://ueaeprints.uea.ac.uk/id/eprint/99388
DOI:

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