Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to p53 is known to occur, but the site of O-GlcNAcylation and its effects on p53 are not understood. Here, we show that Ser 149 of p53 is O-GlcNAcylated and that this modification is associated with decreased phosphorylation of p53 at Thr 155, which is a site that is targeted by the COP9 signalosome, resulting in decreased p53 ubiquitination. Accordingly, O-GlcNAcylation at Ser 149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Our results indicate that the dynamic interplay between O-GlcNAc and O-phosphate modifications coordinately regulate p53 stability and activity.

The transcription factor NFB is activated by phosphorylation and acetylation and plays important roles in inflammatory and immune responses in the cell. Additionally, posttranslational modification of the NFB p65 subunit by O-linked N-acetylglucosamine (OGlcNAc) has been reported, but the modification site of O-GlcNAc on NFB p65 and its exact function have not been elucidated. In this work, we show that O-GlcNAcylation of NFB p65 decreases binding to IB␣ and increases transcriptional activity under hyperglycemic conditions. Also, we demonstrate that both Thr-322 and Thr-352 of NFB p65 can be modified with O-GlcNAc, but modification on Thr-352, not Thr-322, is important for transcriptional activation. Our findings suggest that site-specific O-GlcNAcylation may be a reason why NFB activity increases continuously under hyperglycemic conditions.T ranscription factor NFB plays important roles in inflammatory, immune, and antiapoptotic responses (1-3). In mammals, NFB is present as a dimer composed of various combinations of Rel proteins such as p65 (RelA), RelB, c-Rel, p50/p105, and p52/p100. In most cell types, NFB is composed of p65 and p50 and is localized in the cytosol where it binds inhibitor (IB). Treatment with NFB-activating agents such as tumor necrosis factor ␣ (TNF␣) activates IB kinase (IKK) complexes, inducing phosphorylation in the N terminus of IB. The phosphorylation event induces IB degradation via a ubiquitin-dependent proteolysis. Free NFB translocates to the nucleus and activates the expression of target genes (1, 2).Posttranslational modifications such as phosphorylation (4-9) and acetylation (10, 11) regulate the transcriptional activity of NFB. The activity of NFB is influenced also by the hexosamine biosynthetic pathway, which produces a substrate of O-GlcNAcylation, UDP-GlcNAc (12). Many nucleocytoplasmic proteins are known to be dynamically modified with O-GlcNAc. This modification is modulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) (13-18). O-GlcNAcylation levels play an important role in transcription, translation, nuclear transport, protein stability, and protein-protein interactions and can be increased under hyperglycemic conditions caused by diabetes (16 -18). Although it is known that O-GlcNAcylation of NFB is involved in hyperglycemia-induced NFB activation (12) and is required for lymphocyte activation (19), the specific sites and the function of O-GlcNAcylation on NFB are not well understood.In this work, we show that OGA overexpression downregulates O-GlcNAcylation and inhibits hyperglycemia-induced NFB activation in rat vascular smooth muscle cells (VSMCs). In contrast, up-regulation of O-GlcNAcylation after OGT overexpression and treatment of cells with the O-GlcNAcase inhibitors streptozotocin (STZ) (20) and O-(2-acetamido-2-deoxy-Dglucopyranosylidene)amino-N-phenylcarbamate (PUGNAc)(21) increase NFB transcriptional activity. Additionally, we identify Thr-322 and Thr-352 as O-GlcNAcylation sites in a mutation study and with mass spectrometry analysis. Our data sho...

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO 2 H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO 3 H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H 2 O 2 concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO 3 H) by mass spectrometry. Tsa1p-SO 3 H was not an autoxidation product of Tsa1p-SO 2 H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO 3 H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO 3 H multimer lost its peroxidase activity, it gained ϳ4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO 3 H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO 3 H is a marker of cumulative oxidative stress in cells.