Abstract 1762 Proteasome Inhibition leads to O-GlcNAc-mediated regulation of translation

Following 40 years of broad study, it has been established that O-GlcNAcylation (OGN) functions as a nutrient and stress sensor to regulate enzyme activities, protein localization, protein associations, and protein degradation. OGN cycling is controlled by a sole pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), and over 8,000 protein targets in human cells have been identified thus far. Most recently, emerging data suggests that OGN regulates translation and mRNA selection during nutrient stress. However, the mechanisms by which these processes are regulated by OGN are mostly lacking. Elucidating the molecular mechanisms by which OGN regulates translation is of critical importance in pathogenic conditions including ageing. Our initial studies found that OGT and OGA are tightly associated with ribosomes and translationally active polysomes. Remarkably, when cultured cells (e.g., HEK293F, MEF) were treated with a proteasome inhibitor, we found that the association of both OGT and OGA to ribosomes starkly increased, resulting in elevated OGN of ribosome-associated proteins and translationally active polysomes. Importantly, the dynamic changes of OGT and OGA binding to the polysome differ in response to proteasome inhibition. Multiple techniques were utilized to isolate O-GlcNAcylated proteins from ribosome and polysome fractions. O-GlcNAcomic analysis of the pooled TMT-tagged samples, following proteasome inhibitor treatment (0, 2, 4, 6h), detected 67/80 core ribosome subunits. Newly identified translation elongation factors, E3 ligases, mRNA splicers, and proteasome proteins also displayed significant changes in OGN levels after treatments. The MS/MS data indicate that OGN is involved in translation regulation at various levels while displaying extensive crosstalk with other post-translational modifications, such as ubiquitination, to regulate enzyme/protein activity during the stress response. GalT (Y289L) labeling and WGA pull-down assays confirmed that the chosen target proteins are indeed O-GlcNAcylated. Proteasome and OGN inhibitions also impacted the abundance of many proteins present in the ribosome and polysome fractions. Remarkably, both inducible OGT KO in MEF cells and knockdown of OGT using shRNA in HEK293F cells showed that diminished ribosome OGN nearly abolished ubiquitin binding to ribosome-associated proteins. Polysome profiles showed that OGT KO increases the quantity of non-translational ribosomes independently of MG132 treatment, suggesting that OGN has a role in regulating translation. Our data on the O-GlcNAcome of the translation machinery firmly paves the way for further mechanistic studies. Supported by NIH R01DK61671 and the Georgia Research Alliance. Dr. Hart receives a share of royalty received on sales of the CTD 110.6 antibody, managed by JHU.