AMPK suppression is accompanied by the decreased association of OGT with proteasome.

<p>AMPK suppression in HUVEC either by (A) compound C (10 µmol/L for 2 hour) or by (B) overexpression of AMPK-dominant negative mutant (DN) decreases both the OGT association with proteasome and the O-GlcNAcylation of PA700/S10B (vs. controls), which can be reversed by AICAR pre-incubation (2 mmol/L for 6 hours) in AMPK-present but not AMPK-DN cell. In contrast, constitutive activation of AMPK (overexpression of AMPK-CA vs GFP) (B) increases OGT-PA700 association. All of the blots shown are representative of 3 independent experiments. NS reprents not significant.</p

Increased 26S proteasome activity in AMPK-suppressed HUVEC is correlated with the enhanced association of 19S and 20S sub-complexes.

<p>Compound C (10 µmol/L for 2 hour)-treated HUVEC present (A) AMPK inactivation, (B) an increase in association of PA700/S10B (from 19S complex) with β7 (from 20S complex), which can be blocked by AICAR pre-incubation (2 mmol/L for 6 hours), (C) 26S proteasome assembly (same samples were run on 3–14% native-PAGE under non-reducing condition followed by conventional Western blot on duplicated blots with PA700/S10B and β7 antibodies, respectively), and (D) an increase in 26S proteasome activity (chymotrypsin-like) (n = 3). The increased association of proteasome sub-complex is also observed in (E) HUVEC overexpressing AMPK-DN but not AMPK-CA or GFP. All of the blots shown are representative of 3 independent experiments. NS represents not significant.</p

AMPK depletion is associated with decreased association of OGT with proteasome and increased 26S proteasome assembly and activity in AMPK-KO mice.

<p>Gender (male) and age (12 weeks) matched mice (n = 8/group) with the genotypes of wild type (C57BL/6J) and AMPKα knockout were used. Compared to aortas from wild type (C57BL/6J) mice, (A) aortas from AMPKα knockout mice exhibit (B) a decrease in the association of OGT with proteasome (PA700/S10B), (C) an increase in proteasome assembly (PA700/S10B-β7 association), and (D) an increase in 26S proteasome activity, without alteration in the expression levels of proteasome (β7 or PA700/S10B) or OGT. * represents p<0.05 vs wild type (n = 8).</p

AMPK suppression increases 26S activity in HUVEC.

<p>AMPK suppression in HUVEC either by (A) compound C (10 µmol/L for 2 hour) or by (B) overexpression of AMPK-dominant negative mutant (DN) increases 26S proteasome activities, as demonstrated by ATP-dependent increased chymotrypsin-like, trypsin-like and caspase-like activity on fluorescent proteasome substrates. MG132 (0.5 µmol/L for 1 hour) or AICAR (2 mmol/L for 6 hours) treatment blocks the increased chymotrypsin-like activity in AMPK-suppressed HUVEC. * indicates P<0.05 vs vehicle (DMSO) or GFP, n = 3 per group.</p

Activation of OGT prevents AMPK-inhibition induced 26S proteasome activation.

<p>AMPK suppression by compound C (10 µmol/L for 2 hour) in HUVEC (A) increases the association of PA700/S10B (from 19S complex) with β7 (from 20S complex) accompanied by a decrease of PA700/S10B O-GlcNAc modification, and (B) increases 26S proteasome activity, which can be prevented by pre-incubation of UDP-GlcNAc (25 µmol/L for 30 min), but not by PUGNAc (14 µmol/L for 30 min), the inhibitor of O-GlcNAcase. The blots shown are representative of 3 independent experiments.</p

Loss of OGT increases 26S proteasome assembly.

<p>HUVEC transfected with OGT siRNA but not control siRNA show an increase in (A) association of PA700/S10B (from 19S complex) with β7 (from 20S complex) and (B) 26S proteasome activity, mimicking the effect of AMPK-suppression by compound C (10 µmol/L for 2 hour). The blots shown are representative of 3 independent experiments. * represents p<0.05 vs the control (without compound C treatment).</p

ONOO⁻ nitrates PA700/S10B and increases 26S proteasome activity both <i>in vitro</i> and in intact cell.

<p><i>In vitro</i> (A–C): ONOO⁻ (1 µM) was incubated with the purified 26S proteasome for 5 min; in intact cell (D–E): HUVEC was incubated with ONOO⁻ for 0.5 h, in the presence or absence of uric acid (50 µM pre-incubation for 1 h). Cell free system (<i>in vitro</i>) was subjected to (A) Western blot to detect levels of PA700/S10B and the tyrosine nitration of 26S proteasome/PA700/S10B, (B) 26S proteasome activity (chymotrypsin-like activity), (C) an alternative 26S proteasome activity assay: a substrate-in-gel assay with a fluorogenic substrate followed by fluorescence capturing under the UV light. HUVEC cell lysate was subjected to (D) Western blotting of PA700/S10B tyrosine nitration and (E) assay of 26S proteasome activity (chymotrypsin-like activity). All blots shown are representative of three independent experiments. All results (n = 3) were analyzed with a one-way ANOVA.</p

ONOO⁻ promotes 26S proteasome assembly both <i>in vitro</i> and in intact cell.

<p><i>In vitro</i> (A): ONOO⁻ (1 µM) was incubated with the purified 26S proteasome for 5 min; in intact cell (B): HUVEC was incubated with ONOO⁻ for 0.5 h, in the presence or absence of uric acid (50 µM pre-incubation for 1 h). Cell free system (<i>in vitro</i>) was subjected to (A) separation on a native gradient (3–14%) PAGE gel followed either by Western-blotting (IB) or a direct staining with coomassie brilliant blue (CBB staining) for 26S proteasome assembly. HUVEC cell lysate was subjected to (B) Western blotting of the PA700/S10B co-immunoprecipitates with a β7 antibody. All blots shown are representative of three independent experiments. All results (n = 3) were analyzed with a one-way ANOVA.</p

PA700/S10B tyrosine nitration and 26S proteasome sub-complex association (assembly), but not the PA700/S10B protein levels, are increased in aortic homogenates from mouse models of diabetes, hypertension, and dyslipidemia.

<p>Mouse models of (A) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; Tempol, 1 mmol/kg/drinking water, 2 wks.; n = 5/group); (B) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (C) high fat-diets-induced dyslipidemia (LDLr^−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD and for 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed and their homogenates were subjected to immunoprecipitation and Western blot. The immunoprecipitation assay was performed using either an anti-PA700/S10B or anti-3-NT antibody. All blots shown are representative for mice n = 5. All results were analyzed with a one-way ANOVA. * indicates significant <i>vs.</i> control; NS: not significant <i>vs</i>. control.</p

The 26S proteasome is activated and results in degradation of the target proteins, which can be prevented either by ONOO⁻ inhibition or by MG132 administration, in aortic homogenates from mouse models of diabetes, hypertension, and dyslipidemia.

<p>Mouse models of (A) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; MG132, 5 mg/kg/d, i.p., 2d; n = 5/group); (B) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (C) high fat-diets-induced atherosclerosis (LDLr^−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD, 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed and their homogenates were either subjected to 26S proteasome activity assay (chymotrypsin-like activity) (A–C), or Western blotting with the corresponding antibodies as indicated. All results (n = 5) were analyzed with a one-way ANOVA. * indicates significant <i>vs.</i> control; NS: not significant <i>vs</i>. control.</p