Tyrosine Nitration of PA700 Links Proteasome Activation to Endothelial Dysfunction in Mouse Models with Cardiovascular Risk Factors

Oxidative stress is believed to cause endothelial dysfunction, an early event and a hallmark in cardiovascular diseases (CVD) including hypertension, diabetes, and dyslipidemia. However, the targets for oxidative stress-mediated endothelial dysfunction in CVD have not been completely elucidated. Here we report that 26S proteasome activation by peroxynitrite (ONOO−) is a common pathway for endothelial dysfunction in mouse models of diabetes, hypertension, and dyslipidemia. Endothelial function, assayed by acetylcholine-induced vasorelaxation, was impaired in parallel with significantly increased 26S proteasome activity in aortic homogenates from streptozotocin (STZ)-induced type I diabetic mice, angiotensin-infused hypertensive mice, and high fat-diets -fed LDL receptor knockout (LDLr−/−) mice. The elevated 26S proteasome activities were accompanied by ONOO−-mediated PA700/S10B nitration and increased 26S proteasome assembly and caused accelerated degradation of molecules (such as GTPCH I and thioredoxin) essential to endothelial homeostasis. Pharmacological (administration of MG132) or genetic inhibition (siRNA knockdown of PA700/S10B) of the 26S proteasome blocked the degradation of the vascular protective molecules and ablated endothelial dysfunction induced by diabetes, hypertension, and western diet feeding. Taken together, these results suggest that 26S proteasome activation by ONOO−-induced PA700/S10B tyrosine nitration is a common route for endothelial dysfunction seen in mouse models of hypertension, diabetes, and dyslipidemia

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

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

Inhibition of the 26S proteasome either by ONOO⁻ inhibition or by MG132 administration rescues endothelial dysfunction in mouse models of diabetes, hypertension, and dyslipidemia.

<p>Mouse models of (A/B) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; Tempol, 1 mmol/kg/drinking water, 2 wks.; n = 5/group); (C/E) 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 (D/F) 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 for endothelial function assay. The removed aortas were cut into 3-mm rings, and precontracted with 30 nmol/L of U46619 in organ chambers (PowerLab, ADInstruments, Colorado Springs, Co). (A/C/D) Endothelium-dependent vasodilator responses were determined in the presence of acetylcholine (0.01 to 100 µmol/L). (B/E/F) Endothelium-independent vasodilator responses were determined in the presence of sodium nitroprusside (SNP) (0.0001 to 1 µmol/L). All results were analyzed with a one-way ANOVA. * indicates significant <i>v.s.</i> control; NS: not significant <i>v.s.</i> control.</p

Correction to “Gold Nanoparticles Inhibit Macropinocytosis by Decreasing KRAS Activation”

Correction to “Gold Nanoparticles Inhibit Macropinocytosis by Decreasing KRAS Activation