OGA upregulation prevents the suppression of 26S proteasomes induced by NO.

<p>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.</p

Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2

<div><p>Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co-immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO⁻) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor <em>ex vivo</em> or superoxide scavenger <em>in vivo</em> abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO^–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment.</p> </div

Upregulation of OGA reverses the NO-elevated O-GlcNAcylation of Rpt2.

<p>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.</p

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.</p

NO donors and Bradykinin increase 26S proteasome reporter protein levels and decrease 26S proteasome activity.

<p>The Ub^G76V-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.</p

Identification of Nitric Oxide as an Endogenous Inhibitor of 26S Proteasomes in Vascular Endothelial Cells

<div><p>The 26S proteasome plays a fundamental role in almost all eukaryotic cells, including vascular endothelial cells. However, it remains largely unknown how proteasome functionality is regulated in the vasculature. Endothelial nitric oxide (NO) synthase (eNOS)-derived NO is known to be essential to maintain endothelial homeostasis. The aim of the present study was to establish the connection between endothelial NO and 26S proteasome functionality in vascular endothelial cells. The 26S proteasome reporter protein levels, 26S proteasome activity, and the O-GlcNAcylation of Rpt2, a key subunit of the proteasome regulatory complex, were assayed in 26S proteasome reporter cells, human umbilical vein endothelial cells (HUVEC), and mouse aortic tissues isolated from 26S proteasome reporter and eNOS knockout mice. Like the other selective NO donors, NO derived from activated eNOS (by pharmacological and genetic approach) increased O-GlcNAc modification of Rpt2, reduced proteasome chymotrypsin-like activity, and caused 26S proteasome reporter protein accumulation. Conversely, inactivation of eNOS reversed all the effects. SiRNA knockdown of O-GlcNAc transferase (OGT), the key enzyme that catalyzes protein O-GlcNAcylation, abolished NO-induced effects. Consistently, adenoviral overexpression of O-GlcNAcase (OGA), the enzyme catalyzing the removal of the O-GlcNAc group, mimicked the effects of OGT knockdown. Finally, compared to eNOS wild type aortic tissues, 26S proteasome reporter mice lacking eNOS exhibited elevated 26S proteasome functionality in parallel with decreased Rpt2 O-GlcNAcylation, without changing the levels of Rpt2 protein. In conclusion, the eNOS-derived NO functions as a physiological suppressor of the 26S proteasome in vascular endothelial cells.</p></div

SNP reduces the association of 19S proteasome subcomplex with the 20S subcomplex.

<p>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.</p

A23187 (eNOS activator) and overexpression of eNOS suppresses 26S proteasome functionality in vascular endothelial cells.

<p>The Ub^G76V-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-N^G-Nitroarginine methyl ester.</p

NO increases the O-GlcNAc modification of Rpt2 in endothelial cells.

<p>(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.</p

MGO induces autophagy markers and autophagic flux, whereas genetic or pharmacological induction of autophagy mimics MGO-elicited effects.

<p>A: MGO increased autophagy markers Beclin 1 and LC3B. B: MGO induced LC3 punctae formation (autophagic flux) (Confocal microscopy imaging). C: MGO increased immunoprecipitation of VEGFR2 with LC3B, an autophagy marker and a component of the autophagosome. D: overexpression of Beclin 1 decreased VEGFR2 protein levels, and the reduction was accelerated when MGO was present. E: Rapamycin (1 µM for 1 h) decreased VEGFR2 protein levels. F: Rapamycin (1 µM) reduced endothelial tube formation. BAEC were incubated with MGO (25 µM) for 1 h (A–C) and 16 h (D). Cells were collected for Western blotting with a rabbit derived Beclin-1 or LC 3B antibody and a mouse derived β-actin antibody. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3).</p