GTP cyclohydrolase I phosphorylation and interaction with GTP cyclohydrolase feedback regulatory protein provide novel regulation of endothelial tetrahydrobiopterin and nitric oxide.
Circ Res
; 106(2): 328-36, 2010 Feb 05.
Article
em En
| MEDLINE
| ID: mdl-19926872
ABSTRACT
RATIONALE GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH(4)), an essential cofactor for NO synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH(4) via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH(4) levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH(4) production in response to laminar shear. OBJECTIVE:
We sought to define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. METHODS ANDRESULTS:
Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH(4) levels, and NO production in human endothelial cells. Laminar, but not oscillatory shear stress, caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH(4) levels in vivo.CONCLUSIONS:
These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH(4) to be produced in response to laminar shear stress.
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Base de dados:
MEDLINE
Assunto principal:
Biopterinas
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Células Endoteliais
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Peptídeos e Proteínas de Sinalização Intracelular
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GTP Cicloidrolase
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Óxido Nítrico
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article