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Aspartate Residues Far From the Active Site Drive O-GlcNAc Transferase Substrate Selection

ABSTRACT: Abstract: O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE132205 | GEO | 2019/07/24

REPOSITORIES: GEO

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Project description:The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and OGA activity, localization and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have utilized quantitative proteomics to define OGT’s basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT wild-type, null and hydrogen peroxide treated cell lysates that had been isotopically labeled with light, medium and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture [SILAC]). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation following stress. To validate less-well characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based SILAC approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner.

Project description:Drosophila development is a complex and dynamic process regulated, in part, by members of the Polycomb (Pc), Trithorax (Trx) and Compass chromatin modifier complexes. O-GlcNAc Transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers N-acetyl-D-glucosamine (GlcNAc) onto hydroxyl groups of serine or threonine residues of key transcriptional regulators using the nutrient-derived UDP-GlcNAc as a substrate, which is dynamically removed by O-GlcNAcase (OGA). We performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells and found that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. To examine the in vivo consequences of disturbed O-GlcNAc cycling in the whole animal, we produced a null allele of oga (ogadel.1) in Drosophila. Epigenetic activators including Trx group members Trithorax (Trx), Absent small or homeotic discs 1 (Ash1) and Compass member Set1 histone methyltransferases are O-GlcNAc modified in ogadel.1 mutants. ogadel.1 mutants displayed altered expression of a distinct set of cell cycle related genes in ovaries. Our results suggest that the loss of OGA could affect epigenetic machinery by accumulating O-GlcNAc on numerous chromatin factors including Trx, Ash1 and Set1 in Drosophila. We performed affymetrix tilingarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells to find if that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), ------------------------------- This represents the gene expression component only

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Aspartate Residues Far From the Active Site Drive O-GlcNAc Transferase Substrate Selection

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