The dynamic post-translational modification linked β--acetylglucosamine (-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased GlcNAcylation of numerous proteins, and this increase is thought to promote cell survival. The mechanisms by which theGlcNAc transferase (OGT) and the GlcNAcase (OGA), the enzymes that add and removeGlcNAc, respectively, are regulated during oxidative stress to alter GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevatedGlcNAc levels in U2OS cells but has little impact on the activity of OGT. In contrast, the expression and activity of OGA are enhanced. We hypothesized that this seeming paradox could be explained by proteins that bind to and control the local activity or substrate targeting of OGA, thereby resulting in the observed stress-induced elevations of GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination withtable sotopicabeling of mino acids inell culture (SILAC). This analysis revealed 90 OGA-interacting partners, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase (FAS), filamin-A, heat shock cognate 70-kDa protein, and OGT were confirmed by co-immunoprecipitation. The pool of OGA bound to FAS demonstrated a substantial (∼85%) reduction in specific activity, suggesting that FAS inhibits OGA. Consistent with this observation, FAS overexpression augmented stress-induced GlcNAcylation. Although the mechanism by which FAS sequesters OGA remains unknown, these data suggest that FAS fine-tunes the cell's response to stress and injury by remodeling cellularGlcNAcylation.

The eukaryotic proteome has to be precisely regulated at multiple levels of gene expression, from transcription, translation, and degradation of RNA and protein to adjust to several cellular conditions. Particularly at the translational level, regulation is controlled by a variety of RNA binding proteins, translation and associated factors, numerous enzymes, and by post-translational modifications (PTM). Ubiquitination, a prominent PTM discovered as the signal for protein degradation, has newly emerged as a modulator of protein synthesis by controlling several processes in translation. Advances in proteomics and cryo-electron microscopy have identified ubiquitin modifications of several ribosomal proteins and provided numerous insights on how this modification affects ribosome structure and function. The variety of pathways and functions of translation controlled by ubiquitin are determined by the various enzymes involved in ubiquitin conjugation and removal, by the ubiquitin chain type used, by the target sites of ubiquitination, and by the physiologic signals triggering its accumulation. Current research is now elucidating multiple ubiquitin-mediated mechanisms of translational control, including ribosome biogenesis, ribosome degradation, ribosome-associated protein quality control (RQC), and redox control of translation by ubiquitin (RTU). This review discusses the central role of ubiquitin in modulating the dynamism of the cellular proteome and explores the molecular aspects responsible for the expanding puzzle of ubiquitin signals and functions in translation.Int. J. Mol. Sci. 2020, 21, 1151 2 of 24 initiation, elongation, and termination, with each being fundamental processes conserved across all organisms [20][21][22]. These steps require the proper binding of both RNA and protein factors, and regulation is often facilitated by the alteration of these binding patterns. As ribosomes are highly abundant and responsible for all protein synthesis within the cell, even minor changes to binding patterns could have a large impact on global protein production and cellular health, depending on which steps of translation are altered. Mutations and dysregulation of translational control can lead to a variety of diseases such as cancer, neurological disorders, bone marrow dysfunction and immunodeficiency, among others [23]. As translation is a core process in maintaining cellular function and health, dysfunctional regulation can have devastating results for the cell.Translational control does not occur in a universal manner across a single cell, but varies based on ribosomal subpopulations, with functions specific to cellular localization, transcript targeting, and signaling pathways [24,25]. These subpopulations are distinguishable by RNA and ribosomal protein composition [26,27], binding factors [28], intracellular localization [29,30], and post-translational modifications (PTM) [31]. These subpopulations have a differential capacity to bind and interact with both mRNA and protein factors required for active transla...

O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification that is added and removed from nuclear, cytoplasmic, and mitochondrial proteins by the O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA), respectively. Suggesting that O‐GlcNAc plays a role in cell survival, elevated O‐GlcNAcylation protects cells from many stressors including myocardial ischemia reperfusion injury. To target O‐GlcNAc as a potential cardioprotective therapeutic, we need a better understanding of the mechanisms by which O‐GlcNAc modulates protein function and how OGT and OGA are regulated during times of injury. Recent studies in our laboratory aim to understand the regulation of OGA by its oxidative stress‐dependent binding partners. As existing antibodies do not effectively purify OGA, the goal of this project is to generate and characterize U2OS (human osteosarcoma) and H9C2 (rat myoblast) stable cell lines overexpressing wild type or catalytically dead His6‐tagged OGA. As constitutive overexpression of OGA is toxic, protein expression will be controlled using a tetracycline‐on system (Invitrogen, pT‐Rex). First, we will determine the expression, localization, and activity of His6‐OGA in these cell lines. Ultimately, we will perform a large‐scale isolation of His6‐OGA from oxidatively stressed cells to validate OGA's interactome. These cells lines will be vital for performing physiological studies that will provide molecular insight into the regulation of OGA and the role of O‐GlcNAc in cell survival and cardioprotection.