Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress.

The dynamic post-translational modification O-linked ß-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased O-GlcNAcylation of numerous proteins, and this increase is thought to promote cell survival. The mechanisms by which the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), the enzymes that add and remove O-GlcNAc, respectively, are regulated during oxidative stress to alter O-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevated O-GlcNAc 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 O-GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination with stable isotopic labeling of amino acids in cell 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 O-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 cellular O-GlcNAcylation.

Characterization of tools to detect and enrich human and mouse O-GlcNAcase.

O-linked ß-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.

Identification of Novel Binding Partners for Transcription Factor Emx2.

The mammalian homolog of Drosophila empty spiracles 2 (Emx2) is a homeobox transcription factor that plays central roles in early development of the inner ear, pelvic and shoulder girdles, cerebral cortex, and urogenital organs. The role for Emx2 is best understood within the context of the development of the neocortical region of the cortex, where Emx2 is expressed in a high posterior-medial to low anterior-lateral gradient that regulates the partitioning of the neocortex into different functional fields that perform discrete computational tasks. Despite several lines of evidence demonstrating an Emx2 concentration-dependent mechanism for establishing functional areas within the developing neocortex, little is known about how Emx2 physically carries out this role. Although several binding partners for Emx2 have been identified (including Sp8, eIF4E, and Pbx1), no screens have been used to identify potential protein binding partners for this protein. We utilized a yeast two-hybrid screen using a library constructed from embryonic mouse cDNA in an attempt to identify novel binding partners for Emx2. This initial screen isolated two potential Emx2-binding partner proteins, Cnot6l and QkI-7. These novel Emx2-binding proteins are involved in multiple levels of mRNA metabolism that including splicing, mRNA export, translation, and destruction, thus making them interesting targets for further study.

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis.

O-linked N-acetyl-ß-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-ß-glucosaminidase (O-GlcNAcase). 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 O-GlcNAcylation 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.