C-Terminus of Heat Shock Protein 70–Interacting Protein–Dependent GTP Cyclohydrolase I Degradation in Lambs with Increased Pulmonary Blood Flow

Abstract

We showed that nitric oxide (NO) signaling is decreased in the pulmonary vasculature before the development of endothelial dysfunction in a lamb model of congenital heart disease and increased pulmonary blood flow (Shunt). The elucidation of the molecular mechanism by which this occurs was the purpose of this study. Here, we demonstrate that concentrations of the endogenous NO synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), are elevated, whereas the NOS cofactor tetrahydrobiopterin (BH(4)) is decreased in Shunt lambs. Our previous studies demonstrated that ADMA decreases heat shock protein-90 (Hsp90) chaperone activity, whereas other studies suggest that guanosine-5'-triphosphate cyclohydrolase 1 (GCH1), the rate-limiting enzyme in the generation of BH(4), may be a client protein for Hsp90. Thus, we determined whether increases in ADMA could alter GCH1 protein and activity. Our data demonstrate that ADMA decreased GCH1 protein, but not mRNA concentrations, in pulmonary arterial endothelial cells (PAECs) because of the ubiquitination and proteasome-dependent degradation of GCH1. We also found that Hsp90-GCH1 interactions were reduced, whereas the association of GCH1 with Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) increased in ADMA-exposed PAECs. The overexpression of CHIP potentiated, whereas a CHIP U-box domain mutant attenuated, ADMA-induced GCH1 degradation and reductions in cellular BH(4) concentrations. We also found in vivo that Hsp90/GCH1 interactions are decreased, whereas GCH1-Hsp70 and GCH1-CHIP interactions and GCH1 ubiquitination are increased. Finally, we found that supplementation with l-arginine restored Hsp90-GCH1 interactions and increased both BH(4) and NO(x) concentrations in Shunt lambs. In conclusion, increased concentrations of ADMA can indirectly alter NO signaling through decreased cellular BH(4) concentrations, secondary to the disruption of Hsp90-GCH1 interactions and the CHIP-dependent proteasomal degradation of GCH1.

Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

“…Our previous studies have demonstrated that in Shunt lambs, ADMA levels are increased due to a reduction in DDAH activity (47). Interestingly, this reduction in DDAH activity does not correlate with a reduction in either DDAH1- (Fig.…”

“…The interactions of eNOS/Hsp90 or nitrated CrAT levels were analyzed by immunoprecipitation (IP) analysis as described (47). The efficiency of each IP was normalized by reprobing the membranes with the IP antibody (IP:Hsp90) or by Western blot analysis of 30 lg of the IP protein extract probed using a specific antibody for CrAT (IP:3-nitrotyrosine ).…”

“…Increased ADMA levels are implicated in a number of conditions affecting the cardiovascular system. Our recent studies have shown that the ADMA levels are increased in Shunt lambs secondary to a decrease in dimethylarginine hydrolases (DDAH) activity (47) and that ADMA increases the nitration of mitochondrial proteins in cultured lamb pulmonary arterial endothelial cells (PAEC) (46). Thus, the purpose of this study was to determine whether there was a mechanistic link between increases in ADMA and the disruption of carnitine metabolism; and if so, whether l-arginine supplementation could prevent the mitochondrial dysfunction in Shunt lambs.…”

Hyper-activation of pp60 Src limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury

“…NO and superoxide generation was monitored under conditions that stimulate maximal velocity of the enzyme [28]. The S1177D mutant generates significantly more NO than wildtype eNOS (Fig.…”

Asymmetric Dimethylarginine Induces Endothelial Nitric-oxide Synthase Mitochondrial Redistribution through the Nitration-mediated Activation of Akt1*

“…It is possible that this sequence could interact with the positively charged eNOS mitochondrial translocation loop located at aa 627-631 (RRKRK) (14). Furthermore, we have recently shown in an animal model of pulmonary hypertension associated with increased pulmonary blood flow that increases in ADMA correlates with increased activities of CHIP and Hsp70 (47) and increased translocation of eNOS to the mitochondrion (48). However, further studies will be required to determine whether Hsp70 and/or CHIP play a role in regulating eNOS translocation to the mitochondrion.…”