Over-expression of GTP-cyclohydrolase 1 feedback regulatory protein attenuates LPS and cytokine-stimulated nitric oxide production.
GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate-limiting step for the de novo production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS). The GTP-CH1-BH(4) pathway is emerging as an important regulator in a number of pathologies associated with over-production of nitric oxide (NO) and hence a more detailed understanding of this pathway may lead to novel therapeutic targets for the treatment of certain vascular diseases. GTP-CH1 activity can be inhibited by BH(4) through its protein-protein interactions with GTP-CH1 regulatory protein (GFRP), and transcriptional and post-translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli. However, the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over-expression of GFRP could affect NO production in living cells. Over-expression of N-terminally Myc-tagged recombinant human GFRP in the murine endothelial cell line sEnd 1 resulted in no significant effect on basal BH(4) nor NO levels but significantly attenuated the rise in BH(4) and NO observed following lipopolysaccharide and cytokine stimulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS-mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH(4) and NO levels in vivo.
Nandi
Manasi
M
Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK. m.nandi@ucl.ac.uk
Kelly
Peter
P
Vallance
Patrick
P
Leiper
James
J
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004790:Enzyme Induction; D006136:GTP Cyclohydrolase / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation
C101092:GCHFR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D006136:GTP Cyclohydrolase; C003402:sapropterin