benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and sapropterin

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.

Topics: Animals; Biopterins; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Cysteine Proteinase Inhibitors; Dopamine; Gene Expression Regulation; Isoquinolines; Leupeptins; Mesencephalon; Neurons; PC12 Cells; Phosphorylation; Primary Cell Culture; Proteasome Endopeptidase Complex; Proteolysis; Rats; Receptors, Dopamine D2; Serine; Signal Transduction; Sulfonamides; Tyrosine 3-Monooxygenase; Ubiquitin; Ubiquitination

The ubiquitin-proteasome system has been implicated in oxidative stress-induced endothelial dysfunction in cardiovascular diseases. However, the mechanism by which oxidative stress alters the ubiquitin-proteasome system is poorly defined. The present study was conducted to determine whether oxidative modifications of PA700, a 26S proteasome regulatory subunit, contributes to angiotensin II (Ang II)-induced endothelial dysfunction. Exposure of human umbilical vein endothelial cells to low concentrations of Ang II, but not vehicle, for 6 hours significantly decreased the levels of tetrahydro-l-biopterin (BH4), an essential cofactor of endothelial NO synthase, which was accompanied by a decrease in GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis. In addition, Ang II increased both tyrosine nitration of PA700 and the 26S proteasome activity, which were paralleled by increased coimmunoprecipitation of PA700 and the 20S proteasome. Genetic inhibition of NAD(P)H oxidase or administration of uric acid (a peroxynitrite scavenger) or N(G)-nitro-l-arginine methyl ester (nonselective NO synthase inhibitor) significantly attenuated Ang II-induced PA700 nitration, 26S proteasome activation, and reduction of GTP cyclohydrolase I and BH4. Finally, Ang II infusion in mice decreased the levels of both BH4 and GTP cyclohydrolase I and impaired endothelial-dependent relaxation in isolated aortas, and all of these effects were prevented by the administration of MG132, a potent inhibitor for 26S proteasome. We conclude that Ang II increases tyrosine nitration of PA700 resulting in accelerated GTP cyclohydrolase I degradation, BH4 deficiency, and consequent endothelial dysfunction in hypertension.

Topics: Angiotensin II; Animals; Aorta; Biopterins; Blood Pressure; Cells, Cultured; Cysteine Proteinase Inhibitors; Endothelial Cells; Endothelium, Vascular; GTP Cyclohydrolase; Humans; Hypertension; In Vitro Techniques; Leupeptins; Mice; Mice, Inbred C57BL; Nitrates; Peroxynitrous Acid; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RNA, Small Interfering; Transfection; Tyrosine; Vasodilation

Tetrahydrobiopterin (BH4) deficiency is reported to uncouple the enzymatic activity of endothelial nitric oxide synthase in diabetes mellitus. The mechanism by which diabetes actually leads to BH4 deficiency remains elusive. Here, we demonstrate that diabetes reduced BH4 by increasing 26S proteasome-dependent degradation of guanosine 5'-triphosphate cyclohydrolase I (GTPCH), a rate-limiting enzyme in the synthesis of BH4, in parallel with increased formation of both superoxide and peroxynitrite (ONOO-).. Exposure of human umbilical vein endothelial cells to high glucose concentrations (30 mmol/L D-glucose) but not to high osmotic conditions (25 mmol/L L-glucose plus 5 mmol/L D-glucose) significantly lowered the levels of both GTPCH protein and BH4. In addition, high glucose increased both the 26S proteasome activity and the ubiquitination of GTPCH. Inhibition of the 26S proteasome with either MG132 or PR-11 prevented the high glucose-triggered reduction of GTPCH and BH4. Exposure of human umbilical vein endothelial cells to exogenous ONOO- increased proteasome activity and 3-nitrotyrosine in 26S proteasome. Furthermore, adenoviral overexpression of superoxide dismutase and inhibition of endothelial nitric oxide synthase with N(G)-nitro-L-arginine methyl ester significantly attenuated the high glucose-induced activation of 26S proteasome and the reduction of GTPCH. Finally, administration of MG132 or a superoxide dismutase mimetic, tempol, reversed the diabetes mellitus-induced reduction of GTPCH and BH4 and endothelial dysfunction in streptozotocin-induced diabetes mellitus.. We conclude that diabetes mellitus triggers BH4 deficiency by increasing proteasome-dependent degradation of GTPCH.

Topics: Animals; Antioxidants; Aorta; Biopterins; Cells, Cultured; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; Diabetes Mellitus, Experimental; Endothelial Cells; Glucose; GTP Cyclohydrolase; Guanosine Triphosphate; Leupeptins; Mice; Mice, Inbred C57BL; Nitrogen; Organ Culture Techniques; Peroxynitrous Acid; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reactive Oxygen Species; Spin Labels; Tyrosine; Ubiquitin; Umbilical Veins

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N(G)-nitro-L-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation. Since tetrahydrobiopterin is known to bind near heme and confers stability to the active dimeric structure of nNOS, we wondered if the loss of tetrahydrobiopterin could be an endogenous signal for nNOS ubiquitylation and degradation. We show here in HEK293 cells stably transfected with nNOS that depletion of tetrahydrobiopterin by treatment with 2,4-diamino-6-hydroxypyrimidine leads to destabilization of the dimeric form and enhances ubiquitylation of nNOS. Sepiapterin, a precursor to tetrahydrobiopterin in the salvage pathway, completely reverses the effect of 2,4-diamino-6-hydroxypyrimidine on nNOS ubiquitylation. Consistent with that found in cells, the in vitro ubiquitylation of nNOS by reticulocyte proteins decreases when tetrahydrobiopterin is present. Thus, inadequate amounts of tetrahydrobiopterin may lead to a sustained decrease in the steady state level of nNOS that is not readily reversed.

Topics: Biopterins; Blotting, Western; Cell Line; Chromatography, High Pressure Liquid; Dimerization; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Gene Expression Regulation; Heme; Humans; Hypoxanthines; Immunoprecipitation; Leupeptins; Nitric Oxide Synthase Type I; Pterins; Time Factors; Ubiquitins