guanosine-triphosphate and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

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

In eukaryotic cells, protein kinase CKII is required for progression through the cell division cycle. We recently reported that CKBBP1/SAG/ROC2/Rbx2 associates with the beta-subunit of CKII and is phosphorylated by purified CKII in the presence of ATP in vitro. In this report, we demonstrate that CKBBP1 is efficiently phosphorylated in vitro by purified CKII in the presence of GTP and by heparin-sensitive protein kinase in HeLa cell extract. Mutational analysis indicates that CKII phosphorylates threonine at residue 10 within CKBBP1. Furthermore, CKBBP1 is phosphorylated in vivo and threonine to alanine mutation at residue 10 abrogates the phosphorylation of CKBBP1 observed in vivo, indicating that CKII is a major kinase that is responsible for in vivo phosphorylation of CKBBP1. As compared with the wild-type CKBBP1 or CKBBP1T10E (in which threonine 10 is replaced by glutamate), overexpression of nonphosphorylatable CKBBP1 (CKBBP1T10A) results in accumulation of IkappaBalpha and p27Kip1. Experiments using proteasome inhibitor MG132 and CKII inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole suggest that the accumulation of IkappaBalpha and p27Kip1 results primarily from the reduction of proteasomal degradation in cells expressing CKBBP1T10A, and that CKII-mediated CKBBP1 phosphorylation is required for efficient degradation of IkappaBalpha and p27Kip1. Overexpression of CKBBP1T10A in HeLa cells suppresses cell proliferation and causes accumulation of G1/G0 peak of the cell cycle. Taken together, our results indicate that CKII may control IkappaBalpha and p27Kip1 degradation and thereby G1/S phase transition through the phosphorylation of threonine 10 within CKBBP1.

Topics: Alanine; Casein Kinase II; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Endopeptidases; Enzyme Inhibitors; G1 Phase; Gene Expression; Guanosine Triphosphate; HeLa Cells; Humans; I-kappa B Proteins; Leupeptins; Multienzyme Complexes; Mutagenesis, Site-Directed; NF-KappaB Inhibitor alpha; Phosphorylation; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; RNA; RNA-Binding Proteins; S Phase; Structure-Activity Relationship; Threonine; Transfection; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases