Dataset Information

Quantitative Proteomics Reveals that the OGT Interactome is Remodeled in Response to Oxidative Stress

ABSTRACT: 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.

ORGANISM(S): Mus Musculus

SUBMITTER: Marissa Martinez

PROVIDER: PXD021091 | panorama | Mon Apr 05 00:00:00 BST 2021

REPOSITORIES: PanoramaPublic

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Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress.

Martinez Marissa M Renuse Santosh S Kreimer Simion S O'Meally Robert R Natov Peter P Madugundu Anil K AK Nirujogi Raja Sekhar RS Tahir Raiha R Cole Robert R Pandey Akhilesh A Zachara Natasha E NE

Molecular & cellular proteomics : MCP 20210312

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 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 ...[more]

PMID: 33716169

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Project description:Secondary metabolites play a key role in coordinating ecology and defense strategies of plants. Diversity of these metabolites arise by conjugation of core structures with diverse chemical moieties, such as sugars in glycosylation. Active pools of phytohormones, including those involved in plant stress response are also regulated by glycosylation. While, much is known about the enzymes involved in glycosylation, we know little about their regulation or coordination with other processes. We characterized the flavonoid pathway transcription factor, TRANSPARENT TESTA 8 (TT8) in Arabidopsis thaliana, using an integrative omics strategy. This approach provides a systems level understanding of the cellular machinery that is used to generate metabolite diversity by glycosylation. Metabolomics analysis of TT8 loss-of-function and inducible overexpression lines showed that TT8 coordinates glycosylation of not only flavonoids, but also nucleotides, thus, implicating TT8 in regulating pools of activated nucleotide sugars. Transcriptome and promoter network analyses revealed that TT8 regulome included sugar transporters, proteins involved in sugar binding and sequestration, and a number of carbohydrate active enzymes. Importantly, TT8 affects stress response, along with brassinosteroid and jasmonic acid biosynthesis, by directly binding to the promoters of key genes of these processes. This combined effect on metabolites glycosylation and stress hormones by TT8 inducible overexpression led to significant increase in tolerance towards multiple abiotic and biotic stresses. Conversely, loss of TT8 leads to increased sensitivity to these stresses. Thus, the transcription factor TT8 is an integrator of secondary metabolism and stress response. These findings provide novel approaches to improve broad-spectrum stress tolerance. Gene expression analysis for 6-day old Arabidopsis thaliana seedling were performed for TT8 loss-of-function line, tt8-3 (in Ws background), and its wild type background control, Ws. Two independent biological replicates for each line were hybridized onto two slides of Agilent SurePrint G2 Agilent Arabidopsis V4 (4x44K), with each slide having two technical replicates for each line. Contributors: Amit Rai, Shivshankar Umashankar, Megha, Lim Boon Kiat, Johanan Aow Shao Bing, Sanjay Swarup.

Dataset Information

Quantitative Proteomics Reveals that the OGT Interactome is Remodeled in Response to Oxidative Stress

Publications

Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress.

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