Abstract

O‐linked N‐Acetylglucosamine, more commonly referred to as O‐GlcNAc, is a single glycosidic post‐translational modification. OGT (O‐GlcNAc Transferase) and OGA (O‐GlcNAcase) are the sole enzymes responsible for the addition (O‐GlcNAcylation) and removal of O‐GlcNAc, respectively. O‐GlcNAc, OGA, and OGT are present in all multicellular organisms and organ tissues. Importantly, OGT knockouts are embryonically lethal, demonstrating the requirement for O‐GlcNAcylation in early organismal development. Like most intracellular post‐translational modifications, O‐GlcNAc regulates a number of cellular pathways including mitochondrial respiration, cell cycle progression, and transcription. Recently, we demonstrated that prolonged treatment of SY5Y neuroblastoma cells with Thiamet‐G (TMG), an OGA inhibitor, caused a dramatic reprogramming of the transcriptome. Therefore, we performed next generation RNA sequencing to investigate the transcriptome changes from isolated liver tissues from mice treated with TMG for two weeks. We also performed the same analysis for liver tissue containing an OGT knockout (OGT‐KO) to compare the effects of sustained O‐GlcNAcylation to the loss of O‐GlcNAcylation. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed differences in expression for gene sets between the two groups. Further analysis of the data demonstrated an inverse correlation of transcriptional changes for several gene families between TMG‐treated and OGT‐KO. MAPK/ERK signaling genes were among these families, which we found to be of interest. ERK (extracellular regulated protein kinase) phosphorylates a wide variety of protein substrates, regulating processes such as apoptosis, transcription, and cell cycle progression. OGT‐KO was predicted to down‐regulate this pathway while our previous data predicted TMG to up‐regulate the pathway. To test this observation, we grew HeLa cells in TMG for three weeks and serum starved them overnight. The following day, we returned the serum to the media and harvested the lysate at 0, 5,10, 20, 60, and 120 minute time points. When we performed a western blot analysis of the results, the TMG‐treated group demonstrated dramatic amplification of pERK. This observation demonstrates a novel role for O‐GlcNAc as a regulator of the ERK signaling pathway.Support or Funding InformationNIH NIDDK R01 DK100595This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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