Although the effects of eNOS-derived NO on 26S proteasome functionality are not completely elucidated, the effect of NO on proteasome has drawn increased attention. NO has been found to inhibit the 26S proteasome, resulting in diminished p53 degradation or loss of cell viability. The suppressive effect is PF-4217903 956905-27-4 mediated by S-nitrosylation and reduced proteasomal subunit expression in vascular smooth muscle cells. However, others report that NO enhances proteasome activity and that the activation promoted by NO donor is thought to reduce neutral ceramidase or to protect the endothelial cell from damage induced by H2O2. It is unclear how to reconcile these discrepancies. One plausible solution could be testing the NO-exerted effects in an appropriate 26S proteasome reporter system in intact cells. The 26S proteasome functionality can be regulated through mechanisms dependent and/or independent of proteasomal protease-like activities. Until the first report of imaging 26S proteasome in living cells with a reporter system expressing UbG76V-GFP, assessments of 26S proteasome functionality have predominantly relied on the assay of protease-like activities in whole cell lysates or purified 26S proteasomes. UbG76V-GFP was engineered by expressing a surrogate protein substrate fused with a GDC-0879 Raf inhibitor ubiquitin mutant. The UbG76V mutation is crucial because it makes this GFP-bound ubiquitin resistant to removal by deubiquitinase, which would otherwise prevent the GFP from recognition and degradation by 26S proteasomes. Thus, the UbG76V mutation makes the UbG76V-GFP protein a perfect 26S proteasome substrate. As such, protein levels of GFP represent 26S proteasome functionality in cells. As a surrogate proteasome substrate, UbG76V-GFP was initially designed for the assessment of 26S proteasome inhibition in intact cells or mice. This is because when the 26S proteasome is sufficiently suppressed, the otherwise degraded poly Ub-GFP would accumulate to a significant level for quantification of the GFP fluorescence. Accordingly, UbG76V-GFP mice have been used to monitor proteasome inhibition in models of amyotrophic lateral sclerosis, Alzheimer’s disease, and polyglutamine diseases. By taking the advantage of the UbG76V-GFP reporter, together with an additional modification to its detection, we have been able to monitor the enhancement of 26S proteasome functionality in early diabetes and in glucosetreated cultured cells. This has been accomplished by employing a more sensitive approach that has combined ubiquitin enrichment through ubiquitin affinity binding purification followed by Western blotting of the GFP proteins. Physiological regulation of 26S proteasomes are complex which mechanisms remain incompletely understood; however, it is believed that the mechanisms are multifaceted and include posttranslational modifications. O-GlcNAc modification was the first endogenous inhibitor of the 26S proteasome identified in cells, although the physiological relevance has yet to be established. By utilizing the 26S proteasome reporter system both in cultured cells and mice, the present study sought to identify NO, particularly the eNOS-derived, as an endogenous regulator of the 26S proteasome in vascular endothelial cells and the involvement of proteasome O-GlcNAcylation. With a 26S proteasome reporter system, this study has identified a new mechanism by which NO affects 26S proteasome functionality in vascular endothelial cells. The presented evidence supports an alternative pathway where eNOS-derived NO blocks 
26S proteasome functionality through OGT, the essential enzyme that upregulates protein O-GlcNAc modification. Mechanistically, like NO donors, the eNOS-generated NO increased an OGT-dependent O-GlcNAc modification, likely of Rpt2, one of the subunit of the proteasome regulatory.