Publications (2)8.83 Total impact
-
Article: C-terminus of heat shock protein 70-interacting protein-dependent GTP cyclohydrolase I degradation in lambs with increased pulmonary blood flow.
[show abstract] [hide abstract]
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.American Journal of Respiratory Cell and Molecular Biology 07/2011; 45(1):163-71. · 5.13 Impact Factor -
Article: Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase.
[show abstract] [hide abstract]
ABSTRACT: The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.AJP Heart and Circulatory Physiology 12/2007; 293(5):H3080-7. · 3.71 Impact Factor
