Regulation of tetrahydrobiopterin biosynthesis by shear stress.

An essential cofactor for the endothelial NO synthase is tetrahydrobiopterin (H4B). In the present study, we show that in human endothelial cells, laminar shear stress dramatically increases H4B levels and enzymatic activity of GTP cyclohydrolase (GTPCH)-1, the first step of H4B biosynthesis. In contrast, protein levels of GTPCH-1 were not affected by shear.

Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution.

Tetrahydrobiopterin (BH4) and heat shock protein 90 (hsp90) have been anticipated to regulate endothelial nitric oxide synthase (eNOS)-dependent superoxide anion radical (O2*-) generation in endothelial cells. It is not known, however, whether hsp90 and BH4 increase O2*- in a synergistic manner, or whether this increase is a consequence of downstream changes in eNOS phosphorylation on serine 1179 (eNOS-S1179) and changes in dimer/monomer distribution. Here O2*- production from purified BH4 -free eNOS and eNOS:hsp90 complexes determined by spin-trapping methodology showed that hsp90 neither inhibits O2*- nor alters the requirement of BH4 to inhibit radical release from eNOS.

Changes in tetrahydrobiopterin levels in endothelial cells and adult cardiomyocytes induced by LPS and hydrogen peroxide--a role for GFRP?

Alterations in tetrahydrobiopterin (BH4) levels have significant consequences in vascular pathophysiology. However, the mechanisms regulating BH4 remain poorly understood. The activity of GTP cyclohydrolase I (GTPCH-I), the first enzyme in BH4 biosynthesis, is controlled by protein levels, posttranslational modifications and interaction with GTPCH-I feedback regulatory protein (GFRP).

Cytokine-stimulated GTP cyclohydrolase I expression in endothelial cells requires coordinated activation of nuclear factor-kappaB and Stat1/Stat3.

Endothelial production of nitric oxide (NO) is dependent on adequate cellular levels of tetrahydrobiopterin (BH4), an important cofactor for the nitric oxide synthases. Vascular diseases are often characterized by vessel wall inflammation and cytokine treatment of endothelial cells increases BH4 levels, in part through the induction of GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme for BH4 biosynthesis. However, the molecular mechanisms of cytokine-mediated GTPCH I induction in the endothelium are not entirely clear.

Regulation of tetrahydrobiopterin synthesis and bioavailability in endothelial cells.

Tetrahydrobiopterin (BH4) is a member of the pterin family that has a core structure of pyrazino-2,3-d-pyrimidine rings. Because BH4 is an essential cofactor for the biosynthesis of nitric oxide (a major vasodilator), there is growing interest in BH4 biochemistry in endothelial cells (the cells that line blood vessels). BH4 is synthesized via de novo and salvage pathways from guanosine 5'-triphosphate (GTP) and 7,8-dihydrobiopterin, respectively, in animal cells.

Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein.

GTP cyclohydrolase I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin, a key cofactor necessary for nitric oxide synthase and for the hydroxylases that are involved in the production of catecholamines and serotonin. In animals, the GTPCHI feedback regulatory protein (GFRP) binds GTPCHI to mediate feed-forward activation of GTPCHI activity in the presence of phenylalanine, whereas it induces feedback inhibition of enzyme activity in the presence of biopterin. Here, we have reported the crystal structure of the biopterin-induced inhibitory complex of GTPCHI and GFRP and compared it with the previously reported phenylalanine-induced stimulatory complex.

Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a symmetrical five-membered ring similar to beta-propeller.