Interaction of GTPCH1 with Lys48 linked polyubiquitin chains promotes its degradation.

<p><b>A.</b> HEK293 cells were co-transfected with FLAG-GCH1 and HA tagged ubiquitin plasmids (HA-Ub) for 2 days, cells were lysed, and the proteins were analyzed by immunoblotting using the indicated antibodies. The double plus symbol (<b>++</b>) indicates two folds of HA-Ub plasmids. HEK293 cells were co-transfected with FLAG-GCH1 plasmids and <b>B.</b> Lys48-only ubiquitin plasmids (K48 only-Ub) or, <b>C.</b> Lys63-only ubiquitin plasmids (K63 only-Ub). <b>D.</b> Compare of the GTPCH1 level with or without ubiquitin plasmids overexpression. The blot shown is representative of three independent experiments. Data are shown as mean±standard deviation (n = 3). *^#<i>P</i><0.05 vs. control (without ubiquitin plasmids overexpression). NS indicates <i>P</i>>0.05 vs. control.</p

GTPCH1 binds polyubiquitin in cultured cells.

<p><b>A.</b> Endogenous GTPCH1 binds polyubiquitin in mouse endothelial cells (SVEC4-10). Mouse endothelial cells were grown to confluence and cell lysates were incubated with UIM-agarose for ubiquitin affinity precipitation (AP). The interaction of GTPCH1 with polyubiquitin chains was analyzed by immunoblotting. The black triangle (▸) indicates the bound GTPCH1 by polyubiquitin chains. Control beads without UIM were used as a negative control. <b>B.</b> Co-immunoprecipitation of GTPCH1 with polyubiquitin in GTPCH1 overexpressed HEK293 cells. FLAG-GCH1 plasmids or empty vectors were transfected into HEK293 cells for 2 days. Cell lysates were incubated with the ubiquitin antibody and immunoprecipitated using protein A/G beads. The asterisk (*) indicates IgG bands. HEK293 transfected with empty vectors were used as a negative control. <b>C.</b> Polyubiquitin chains were co-immunoprecipitated with GTPCH1. HEK293 cell lysates expressing FLAG-GTPCH1 or control vector were incubated with anti-FLAG resin. Proteins that co-immunoprecipitated with FLAG-GTPCH1 were analyzed by immunoblotting using the indicated antibodies. The blot shown is representative of five independent experiments.</p

Inhibition of the 26S proteasome causes the accumulation of the GTPCH1/Ub complex.

<p><b>A and B.</b> HEK293 cells were transfected with FLAG-GCH1 or empty vector (control) for 2 days and treated with the 26S proteasome inhibitors MG132 or lactacystin (Lac, irreversible inhibitor) for 6 hours. Lysates from cells expressing FLAG-GTPCH1 were immunoprecipitated with anti-FLAG resin and immunoblotted using the indicated antibodies. HEK293 transfected with empty vectors were used as a negative control. The asterisk (*) indicates IgG bands. <b>C.</b> HEK293 cells were co-transfected with FLAG-GCH1 and HA-Ub plasmids and treated with or without MG132 for 6 hours. Lysates from cells expressing FLAG-GTPCH1 were immunoprecipitated with anti-FLAG resin and immunoblotted with the FLAG, HA, or β-actin antibodies. The blot shown is representative of three independent experiments.</p

GTPCH1 non-covalently interacts with polyubiquitin in mouse heart and lung <i>in vivo</i>.

<p>Mice tissues (n = 4) were isolated and the homogenates were generated as described in Experimental Procedures. The tissue lysates were incubated with UIM-agarose for ubiquitin affinity precipitation (AP). The proteins that interacted with polyubiquitin chains were analyzed by western blot using specific antibodies. <b>A.</b> GTPCH1 and polyubiquitin chains interaction in mouse heart and <b>B.</b> mouse lung. Control beads without UIM were used as a negative control are included in all experiments. The black triangle (▸) indicates the bound GTPCH1 by polyubiquitin chains and the asterisk (*) indicates a non-specific band. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043306#s3" target="_blank">Results</a> were obtained from four mice.</p

The ubiquitin-binding domain (UBD) of GTPCH1 mediates the interaction with ubiquitin chains.

<p><b>A</b>. Alignment of ubiquitin binding domains found in multiple proteins. The accession numbers are for GenBank (gi) and residue numbers of the sequences are shown on the left. Below, (*) indicates conserved hydrophobic core residues and (#) indicates conserved “GFP-loop”. Abbreviations used: HS, human; SC, yeast; MM, mouse. <b>B</b>. Schematic of the GTPCH1 deletion mutants. <b>C & D</b>. GST-tagged GTPCH1 (42–250) or deletion mutants (NΔ1∼NΔ3) were expressed in <i>E. coli</i>. Purified GST-GTPCH1 proteins bound to GSH beads were incubated with the synthesized tetraubiquitin chains (Ub4). The bound proteins were detected by immunoblot using the ubiquitin antibody. GST alone was used as a negative control. The blot shown is representative of three independent experiments. Data are shown as mean±standard deviation (n = 3). *<i>P</i><0.05 vs. control. NS indicates <i>P</i>>0.05 vs. control.</p

Induction of HO-1 protein and AMPK activation by OA-NO₂.

<p><b>A</b>) BAECs were incubated with OA-NO₂ at the indicated concentrations or with BSA (vehicle) for 16 h, and western blot analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a> to detect HO-1 protein expression and AMPK phosphorylation at Thr172. The blot is representative of those obtained from three separate experiments. Corresponding densitometric analyses of phosphorylated AMPK and ACC are shown. *<i>p</i><0.05 <i>vs.</i> control. <b>B</b>) BAECs were incubated with 2.5 µM OA-NO₂ for the indicated times, and western blotting was performed as above. The blot is representative of three blots obtained from three separate experiments. *<i>p</i><0.05 <i>vs.</i> corresponding control. <b>C</b>) Confluent BAECs were exposed to vehicle or OA-NO2 (2.5 µM) for 16 h. AMPKα was immunoprecipitated from cell lysates (1 mg) with a specific antibody. AMPK activity was assayed by ³²P-ATP incorporation into the SAMS peptide. *<i>p</i><0.05 <i>vs.</i> control. <b>D</b>) BAECs were incubated with the indicated concentrations of OA for 16 h. Western blotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a>. <b>E</b>) BAECs were infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). Infected and non-infected cells were treated with 2.5 µM OA-NO₂ for 16 h. AICAR and metformin were used as positive controls. The blot is representative of three blots obtained from three separate experiments.</p

Activation of AMPK by OA-NO₂ does not require LKB1.

<p><b>A</b>) Phosphorylation of LKB1 Ser428 was not affected by OA-NO₂ in BAECs. Confluent BAECs were exposed to 2.5 µM OA-NO₂ for 16 h, and phosphorylated LKB1-Ser428 was detected by a phospho-specific antibody in western blots. The blot is a representative of three blots obtained from three independent experiments. <i>Lower panels</i>: summary data (<i>n</i> = 3). <b>B</b>) LKB1 is not required for AMPK activation by OA-NO₂. Confluent LKB1-deficient Hela-S3 cells were exposed to 2.5 µM OA-NO₂ for 16 h, and then AMPK and ACC phosphorylation were assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031056#s4" target="_blank">Materials and Methods</a>. The blot is representative of three blots obtained from three independent experiments. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; <i>n</i> = 3). <b>C</b>) LKB1 siRNA did not abolish OA-NO₂-stimulated AMPK activation in HUVECs. HUVECs were incubated with LKB1-specific siRNA or control siRNA for 48 h and then treated with OA-NO₂ or vehicle for 16 h. After treatment, cell lysates were analyzed for LKB1 protein levels and AMPK phosphorylation at Thr172. <i>Lower panels</i>: summary data (*<i>p</i><0.05 <i>vs.</i> control; <i>n</i> = 3).</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 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