O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a ubiquitous and dynamic post-translational modification known to modify over 3,000 nuclear, cytoplasmic, and mitochondrial eukaryotic proteins. Addition of O-GlcNAc to proteins is catalyzed by the O-GlcNAc transferase and is removed by a neutral-N-acetyl-β-glucosaminidase (OGlcNAcase). O-GlcNAc is thought to regulate proteins in a manner analogous to protein phosphorylation, and the cycling of this carbohydrate modification regulates many cellular functions such as the cellular stress response. Diverse forms of cellular stress and tissue injury result in enhanced O-GlcNAc modification, or O-GlcNAcylation, of numerous intracellular proteins. Stress-induced OGlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, calcium homeostasis, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation. Here, we will discuss the regulation of these processes by O-GlcNAc and the impact of such regulation on survival in models of ischemia reperfusion injury and trauma hemorrhage. We will also discuss the misregulation of O-GlcNAc in diseases commonly associated with the stress response, namely Alzheimer's and Parkinson's diseases. Finally, we will highlight recent advancements in the tools and technologies used to study the O-GlcNAc modification.

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.

Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) posttranslational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.

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.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential regulatory post-translational modification of thousands of nuclear, cytoplasmic, and mitochondrial proteins. O-GlcNAc is dynamically added and removed from proteins by the O-GlcNAc transferase and the O-GlcNAcase (OGA), respectively. Dysregulation of O-GlcNAc-cycling is implicated in the etiology of numerous diseases including tumorigenesis, metabolic dysfunction, and neurodegeneration. To facilitate studies focused on the role of O-GlcNAc and OGA in disease, we sought to identify commercially available antibodies that enable the enrichment of full-length OGA from lysates of mouse and human origin. Here, we report that antibodies from Abcam and Bethyl Laboratories can be used to immunoprecipitate OGA to near-saturation from human and mouse cell lysates. However, Western blotting analysis indicates that both antibodies, as well as three non-commercially available antibodies (345, 346, 352), detect full-length OGA and numerous cross-reacting proteins. These non-specific signals migrate similarly to full-length OGA and are detected robustly, suggesting that the use of appropriate controls is essential to avoid the misidentification of OGA.

O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification of nuclear, cytoplasmic, and mitochondrial proteins. While O‐GlcNAc has been demonstrated to modulate numerous cellular processes, the molecular mechanisms by which O‐GlcNAc mediates protein function are not well defined. One mechanism by which O‐GlcNAc may regulate protein function is by promoting protein‐protein interactions. In this study we have investigated whether the intracellular hyaluronan‐binding protein p32 binds proteins in an O‐GlcNAc‐dependent manner. Using ELISA and dot blotting techniques we have demonstrated that p32 binds GlcNAc‐conjugates preferentially to other sugar‐conjugates in vitro. Suggesting that p32 interacts with O‐GlcNAc in vivo, p32 enriches O‐GlcNAc‐modified proteins similar to the O‐GlcNAc‐specific antibody RL2. Finally, our data demonstrate that overexpression of p32 fused to the biotin ligase BirA results in a biotinylation pattern that can be modulated by manipulating O‐GlcNAc levels. Preliminary studies suggest that one candidate protein bound by p32 in an O‐GlcNAc‐dependent manner is the O‐GlcNAc transferase (OGT), the enzyme responsible for the addition of O‐GlcNAc. Taken together, these data suggest that p32 may contain an O‐GlcNAc‐binding motif that dictates its ability to interact with a subset of intracellular proteins such as OGT. Grant Funding Source: Supported by the National Heart, Lung, and Blood Institute (P01HL107153)

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Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) post-translational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated for the first time that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs. Citation Format: Takumi Shiraishi, Phuoc T. Tran, Reem Malek, Audrey Lafargue, Mustafa Barbhuiya, Xing Wang, Brian Simons, Matthew Ballew, Katriana Nugent, Jennifer Groves, Russell Williams, Hailun Wang, James Verdone, Gokben Yildirir, Roger Henry, Bin Zhang, John Wong, Ken Wang, Barry Nelkin, Kenneth Pienta, Dean Felsher, Natasha Zachara, Kekoa Taparra. O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B11.