title: Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Prevents Dihydrofolate Reductase Degradation via Promoting S-Nitrosylation.
abstract: OBJECTIVE
  Dihydrofolate reductase (DHFR) is a key protein involved in tetrahydrobiopterin (BH4) regeneration from 7,8-dihydrobiopterin (BH2). Dysfunctional DHFR may induce endothelial nitric oxide (NO) synthase (eNOS) uncoupling resulting in enzyme production of superoxide anions instead of NO. The mechanism by which DHFR is regulated is unknown. Here, we investigate whether eNOS-derived NO maintains DHFR stability.
  
  
  RESULTS
  DHFR activity, BH4 content, eNOS activity, and S-nitrosylation were assessed in human umbilical vein endothelial cells and in aortas isolated from wild-type and eNOS knockout mice. In human umbilical vein endothelial cells, depletion of intracellular NO by transfection with eNOS-specific siRNA or by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-both of which had no effect on DHFR mRNA levels-markedly reduced DHFR protein levels in parallel with increased DHFR polyubiquitination. Supplementation of S-nitroso-l-glutathione (GSNO), a NO donor, or MG132, a potent inhibitor of the 26S proteasome, prevented eNOS silencing and PTIO-induced DHFR reduction in human umbilical vein endothelial cells. PTIO suppressed S-nitrosylation of DHFR, whereas GSNO promoted DHFR S-nitrosylation. Mutational analysis confirmed that cysteine 7 of DHFR was S-nitrosylated. Cysteine 7 S-nitrosylation stabilized DHFR from ubiquitination and degradation. Experiments performed in aortas confirmed that PTIO or eNOS deficiency reduces endothelial DHFR, which can be abolished by MG132 supplementation.
  
  
  CONCLUSIONS
  We conclude that S-nitrosylation of DHFR at cysteine 7 by eNOS-derived NO is crucial for DHFR stability. We also conclude that NO-induced stabilization of DHFR prevents eNOS uncoupling via regeneration of BH4, an essential eNOS cofactor.
authors.affiliation: From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Q.L., Y.D., Q.W., L.X., P.S., M.-H.Z.); and Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Zhejiang, China (Z.C.).
authors.forename: Zhejun
authors.initials: Z
authors.lastname: Cai
authors.affiliation: From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Q.L., Y.D., Q.W., L.X., P.S., M.-H.Z.); and Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Zhejiang, China (Z.C.).
authors.forename: Qiulun
authors.initials: Q
authors.lastname: Lu
authors.affiliation: From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Q.L., Y.D., Q.W., L.X., P.S., M.-H.Z.); and Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Zhejiang, China (Z.C.).
authors.forename: Ye
authors.initials: Y
authors.lastname: Ding
authors.affiliation: From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Q.L., Y.D., Q.W., L.X., P.S., M.-H.Z.); and Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Zhejiang, China (Z.C.).
authors.forename: Qilong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Q.L., Y.D., Q.W., L.X., P.S., M.-H.Z.); and Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Zhejiang, China (Z.C.).
authors.forename: Lei
authors.initials: L
authors.lastname: Xiao
authors.affiliation: From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Q.L., Y.D., Q.W., L.X., P.S., M.-H.Z.); and Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Zhejiang, China (Z.C.).
authors.forename: Ping
authors.initials: P
authors.lastname: Song
authors.affiliation: From the Center for Molecular and Translational Medicine, Georgia State University, Atlanta (Z.C., Q.L., Y.D., Q.W., L.X., P.S., M.-H.Z.); and Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Zhejiang, China (Z.C.). mzou@gsu.edu.
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Zou
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology*; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004795:Enzyme Stability; D016166:Free Radical Scavengers / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D034622:RNA Interference; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D054875:Ubiquitination
chemical_list: D016166:Free Radical Scavengers; D061988:Proteasome Inhibitors; D001708:Biopterins; D009569:Nitric Oxide; C017226:7,8-dihydrobiopterin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D013762:Tetrahydrofolate Dehydrogenase; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D003545:Cysteine
keywords: Nos3 protein; human umbilical vein endothelial cells; mouse; nitric oxide; sapropterin; tetrahydrofolate dehydrogenase