Hence, O-GlcNAc modification is considered an endogenous inhibitor of the 26S proteasome and O-GlcNAc modification connects a nutritional sensor to proteasome functional regulation. In line with these findings, we found that OGT is essential in mediating NO-dependent 26S proteasome suppression both in cultured cells and aortas of the eNOS-KO mice, likely through the control of Rpt2 O-GlcNAcylation. Conversely, suppression of OGT or overexpression of OGA prevented the NO-mediated effects on 26S proteasomes. OGT appears to be crucial for this event, since NO-mediated 26S proteasome inhibition is blocked by siRNAmediated OGT downregulation. The most conclusive evidence that eNOS-derived NO suppresses the 26S proteasome comes from analysis of aortas from eNOS-/- mice. We found that, compared to WT aortic tissues, eNOS2/2 mice exhibit elevated 26S proteasome activity in parallel with decreased Rpt2 OGlcNAcylation. All these data suggest the axis of NO-OGT/ OGA-Rpt2-O-GlcNAcylation is truly associated with the regulation of 26S proteasome functionality. Yet, the decisive role for OGlcNAc modification of Rpt2 in this axis has yet to be established, because other subunits of proteasome, such as 20S subcomplex, are potentially targets of O-GlcNAcylation. Nevertheless, the present study supports the notion that eNOS-derived NO, an endothelial protective molecule at basal low concentration, maintains a basal low 26S proteasome functionality, which is achieved possibly through keeping Rpt2 O-GlcNAcylated and in turn, keeping the 26S proteasome at minimal levels of functionality in endothelial cells. The next novel aspect of the study was the generation of 26S proteasome reporter mice with eNOS deleted. With this approach, an Dabrafenib supply intrinsic inhibitory role of eNOS on 26S proteasome functionality was uncovered in whole animal in vivo in the present study. Interestingly, eNOS, the enzyme that maintains the basal and physiological levels of NO in endothelial cells, can be a target of proteasome. Thus, the endogenous inhibitory potential of NO to 26S proteasomes could be a line of self-defense against degradation so that eNOS remains functionally active, although long term inhibition of proteasome generates the opposite effects. The identification of such a role for NO may add an alternative mechanism to peroxynitrite-mediated 26S proteasome activation in animal models in vivo, since formation of peroxynitrite in the presence of excessive superoxide will Gefitinib dramatically reduce NO bioavailability, thus compromise the suppressive impact of NO on proteasome. The generations of proteasome reporter eNOS-knockout mice will also help to identify factor that relates to OGT and mediates NO-dependent 26S proteasome regulation in vasculature. It remains obscure how eNOS-derived NO affects OGT in endothelial cells. OGT activity can be regulated at several levels, including transcription, splicing, translation, protein stability, and post-translational modifications. Among them, S-nitrosylation is likely the mechanism that associates with a direct impact from NO, although higher concentrations of NO may be required for S-nitrosylation. Recently, S-nitrosylation was shown to suppress OGT, because the removal of the nitrosyl group activated OGT in polysaccharide-treated macrophages. Such an inhibitory mechanism could not explain the indispensable role of OGT in NO-elicited effects, suggesting that other mechanism would apply. HSP90 seems involved in OGT turnover because inactivation of HSP90 reduces OGT protein stability. As a client protein of HSP90, eNOS is positively regulated by HSP90 through strong interaction, although the interaction may inactivate HSP90, likely serving as a negative feedback mechanism by NO.