Figure 1.
NO donors and Bradykinin increase 26S proteasome reporter protein levels and decrease 26S proteasome activity.
The UbG76V-GFP-expressing cells were incubated with (A) MG132 (0.5 µM), or vehicle (DMSO, 0.5%) for 24 h followed by Western blotting of the 26S proteasome reporter protein poly-Ub-GFP by using a rabbit-derived anti-GFP antibody, with or without GFP enrichment by agarose bead conjugated with an anti-GFP antibody. The same type of cells were also incubated with SNP (50 µM), DETA-NONOate (50 µM), or Bradykinin (1 µM) for indicated time up to 4 h. The cell lysate were subjected to (B) Western blotting of reporter proteins with an anti-GFP antibody and to (C) 26S proteasome activity assay by measuring the chymotrypsin-like activity of the cell lysates. The shown blots were representative of at least 3 independent experiments with similar results. * represent p<0.05 vs control (0 h) (n = 3), otherwise, not significant. An overlaid portion (less exposure) of each whole blot in (B) indicating Ub-GFP is presented. DETA-NONOate, Diethylenetriamine NONOate; DMSO, dimethyl sulfoxide; SNP, sodium nitroprusside; Ub-GFP, ubiquitin-green fluorescent protein.
Figure 2.
SNP reduces the association of 19S proteasome subcomplex with the 20S subcomplex.
The HUVEC were incubated with (A) SNP (50 µM) for 0.5 h or (B) DETA-NONOate (10 µM) for 0.5 h followed by isolation of intact 26S proteasomes which were then subjected to immunoprecipitation using agarose immobilized with antibody recognizing the 20S proteasome subcomplex (Enzo Life Sciences, Farmingdale, NY; Cat# BML-PW1075). Rpt2 was detected via Western blotting on the immunoprecipitates of 20S proteasome subcomplex. Purification of intact 26S proteasomes was conducted according to the instruction of the manufacture (UBPBio, Aurora, CO; Rapid 26S Proteasome Purification Kit; Cat# J4320). p<0.05 vs control (n = 3). Ctrl, control; SNP, sodium nitroprusside.
Figure 3.
A23187 (eNOS activator) and overexpression of eNOS suppresses 26S proteasome functionality in vascular endothelial cells.
The UbG76V-GFP-expressing HUVEC were incubated with (A) A23187 (1 µM) for indicated time up to 4 h and the 26S proteasome reporter protein poly-Ub-GFP was stained with a rabbit-derived anti-GFP antibody, total eNOS and p-eNOS (Ser 1177) was stained with respective antibodies through Western blot. (B) Assay of the chymotrypsin-like activity. (C) Preincubation of L-NAME (1 mM) abolished A23187 enhanced reporter protein accumulation. (D) The chymotrypsin-like activity. (E) Adenoviral overexpression eNOS increased the 26S proteasome reporter protein poly-Ub-GFP, which was prevented by preincubation of L-NAME (1 mM) for 1 h. (F) The chymotrypsin-like activity. The presented blots were representative of at least 3 independent experiments with similar results. * represent p<0.05 vs control or 0 h (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ad-, Adenoviral overexpression; NS, not significant (v.s. control); Ub-GFP, ubiquitin-green fluorescent protein; eNOS, endothelial nitric oxide synthase; L-NAME, L-NG-Nitroarginine methyl ester.
Figure 4.
NO enhances the immunological staining of the 26S proteasome reporter proteins in endothelial cells.
UbG76V-GFP-expressing HUVEC were respectively treated with control (vehicle buffer), SNP, Bradykinin, and A23187 for 1h and subjected to immunofluorescent staining with a commercial immune-staining kit including ProLong Gold and SlowFade Gold Antifade for GFP (with a primary anti-GFP antibody) or DAPI and a secondary antibody conjugated with a red fluorescent dye, Alex Fluor 594 at (A) low and (B) high-power fields of the same slides. All images shown are representative of three independent experiments. (C) Quantification results of (B). * represent p<0.05 vs control (n = 3). Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; SNP, sodium nitroprusside; Ub-GFP, ubiquitin-green fluorescent protein.
Figure 5.
NO at high concentrations inhibits the activity of purified 26S proteasomes.
(A) SNP decreased the chymotrypsin-like activity of the purified 26S proteasome. (B) DETA-NONOate reduced the chymotrypsin-like activity of the purified 26S proteasome. The purified 26S proteasome (Enzo Life Sciences, Inc) were incubated with SNP or DETA-NONOate at indicated concentrations up to 1000 µM for 0.5 h followed by assay of the chymotrypsin-like activity. * represent p<0.05 vs control (n = 3). DETA-NONOate, Diethylenetriamine NONOate; SNP, sodium nitroprusside.
Figure 6.
NO increases the O-GlcNAc modification of Rpt2 in endothelial cells.
(A) HUVEC respectively treated with A23187, Bradykinin, and SNP presented increased O-GlcNAc modification of Rpt2 without altering total Rpt2 protein levels. (B) Overexpression of eNOS but not GFP upregulated Rpt2 O-GlcNAcylation without changing the levels of total Rpt2 protein. Rpt2 O-GlcNAcylation was detected with the WGA protocol. (C) Confirming Rpt2-GlcNAcylation by repeating experiments in (A) but using an O-GlcNAc antibody (CD110.6) to pull down O-GlcNAcylated proteins. (D) Confirming the effect of NO on Rpt2-GlcNAcylation by repeating experiments in (C) but with DETA-NONOate. The shown blots were representative of at least 3 independent experiments with similar results. * represent p<0.05 vs control (n = 3), otherwise, not significant. Ad-, Adenoviral overexpression; Ctrl, control; SNP, sodium nitroprusside; WGA, wheat germ agglutinin; GFP, green fluorescent protein; eNOS, endothelial nitric oxide synthase.
Figure 7.
OGT mediates NO-induced suppression of 26S proteasome functionality.
OGT siRNA knockdown abolished the proteasome reporter protein accumulation and Rpt2 O-GlcNAcylation induced by (A) SNP; (B) Bradykinin; and (C) A23187. Rpt2 O-GlcNAcylation was detected with the WGA protocol. The shown blots were representative of at least 3 independent experiments with similar results. OGT knockdown by siRNA restored 26S proteasome activity in cell treated with (D) SNP; (E) Bradykinin; and (F) A23187. 26S proteasome activity was quantified by measuring chymotrypsin-like activity in the cell lysates. * represent p<0.05 vs control (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ctrl, control (scrambled) siRNA; NS, not significant (v.s. control); OGT, O-GlcNAc transferase; SNP, sodium nitroprusside; Ub-GFP, ubiquitin-green fluorescent protein; WGA, wheat germ agglutinin.
Figure 8.
Upregulation of either O-GlcNAcylation or OGT suppresses 26S proteasome functionality.
The UbG76V-GFP-transfected HUVEC were treated with vehicle (medium) and glucosamine (5 mM) for indicated time up to 4h and followed by (A) quantifications of poly-Ub-GFP protein levels with an anti-GFP antibody and O-GlcNAc modification of Rpt2 with an anti-Rpt2 antibody (on WGA pull-down) in Western blot; (B) Western blotting of global O-GlcNAc modified proteins with an anti-O-GlcNAc antibody; (C) proteasomal chymotrypsin-like activity assay. (D) Overexpression of OGT not GFP increased both Rpt2 O-GlcNAcylation and the levels of reporter protein. (E) Chymotrypsin-like activity in GFP- and OGT-overexpressing cells. The blots shown were representative of 3 independent experiments with similar results. * represents p<0.05 vs the control (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ub-GFP, ubiquitin-green fluorescent protein; OGT, O-GlcNAc transferase; UDP-GlcNAc, Uridine diphosphate-GlcNAc; WGA, wheat germ agglutinin.
Figure 9.
Overexpression of OGA mimics the effects of OGT knockdown by siRNA on 26S proteasomes.
(A) UbG76V-GFP-expressing HUVEC were transfected either with control or OGA siRNA and cell lysates were subjected to Western blot to detect reporter protein levels and Rpt2 O-GlcNAcylation. (B) chymotrypsin-like activity in siRNA treated cells. (C) Adenoviral overexpression of OGA, not GFP, decreased both the levels of reporter protein and Rpt2 O-GlcNAcylation. (D) chymotrypsin-like activity in adenovirus infected cells. The blots shown were representative of 3 independent experiments with similar results. * represents p<0.05 vs the control (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ctrl, control (scrambled) siRNA; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; Ub-GFP, ubiquitin-green fluorescent protein; WGA, wheat germ agglutinin.
Figure 10.
Upregulation of OGA reverses the NO-elevated O-GlcNAcylation of Rpt2.
Adenoviral overexpression of OGA abolished Rpt2 O-GlcNAcylation induced by (A) SNP; (B) Bradykinin; (C) A23187; and (D) overexpression of eNOS. Rpt2 O-GlcNAcylation was detected with the WGA protocol. The shown blots were representative of at least 3 independent experiments with similar results. Ad-, Adenoviral overexpression; eNOS, endothelial nitric oxide synthase; NS, not significant (v.s. control); OGA, O-GlcNAcase; SNP, sodium nitroprusside; WGA, wheat germ agglutinin.
Figure 11.
OGA upregulation prevents the suppression of 26S proteasomes induced by NO.
Adenoviral overexpression of OGA prevented the proteasome reporter protein accumulation induced by (A) SNP; (B) Bradykinin; and (C) A23187. GOA upregulation via overexpression restored 26S proteasome activity which was otherwise inhibited by (D) SNP; (E) Bradykinin; and (F) A23187. 26S proteasome activity was quantified by measuring chymotrypsin-like activity in the cell lysates. * represent p<0.05 vs control (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ad-, Adenoviral overexpression; NS, not significant (v.s. control); OGA, O-GlcNAcase; SNP, sodium nitroprusside; Ub-GFP, ubiquitin-green fluorescent protein.
Figure 12.
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.
(A) Genotyping of wild type (C57BL/6J) mice, UbG76V-GFP (eNSO wild type) mice, and UbG76V-GFP mice lacking eNOS by PCR analysis. (B) Gender (male) and age (12 weeks) matched wild type mice, UbG76V-GFP/eNOS+/+ mice, and UbG76V-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.