Loss of eNOS enhances 26S proteasome functionality which is associated with a reduction in Rpt2 O-GlcNAcylation and an increase in proteasome chymotrypsin-like activity.

<p>(A) Genotyping of wild type (C57BL/6J) mice, Ub^G76V-GFP (eNSO wild type) mice, and Ub^G76V-GFP mice lacking eNOS by PCR analysis. (B) Gender (male) and age (12 weeks) matched wild type mice, Ub^G76V-GFP/eNOS^+/+ mice, and Ub^G76V-GFP/eNOS^−/− mice (n = 5/group) were used. The 26S proteasome reporter protein poly-Ub-GFP was stained with a rabbit-derived anti-GFP antibody through Western blot. The eNOS protein of the aortic tissues was detectable in eNOS^+/+ but not eNOS^−/− mice. (C) Aortic tissues of the eNOS^−/− vs WT mice exhibited a decrease in Rpt2 O-GlcNAcylation, without changing the protein levels total Rpt2 and β7. (D) The 26S proteasome activity (chymotrypsin-like activity) was significantly higher in eNOS^−/− vs WT mouse aortic tissues. (E) Proposed mechanisms of 26S proteasome regulation by eNOS-derived NO in vascular endothelial cells. In vascular endothelial cells, the eNOS-derived NO, which has been known to be expressed constitutively at low basal levels, maintains basal functionality of the 26S proteasome. This was achieved through an OGT-dependent O-GlcNAc modification of proteasome, likely on Rpt2, a key subunit of the proteasome regulatory complex recognized for this type of modification and associated with 26S proteasome function. The supporting evidence was obtained through either genetic or pharmacologic approaches both in 26S proteasome reporter cell and mouse models. eNOS, endothelial nitric oxide synthase; OGT, O-GlcNAc transferase; SNP, sodium nitroprusside; WGA, wheat germ agglutinin; WT, wild type.