title: Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.
abstract: BACKGROUND
  Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.
  
  
  METHODS
  Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.
  
  
  RESULTS
  Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.
  
  
  CONCLUSIONS
  Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.
authors.affiliation: Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Panjamaporn
authors.initials: P
authors.lastname: Sangwung
authors.forename: Todd M
authors.initials: TM
authors.lastname: Greco
authors.forename: Yanzhuang
authors.initials: Y
authors.lastname: Wang
authors.forename: Harry
authors.initials: H
authors.lastname: Ischiropoulos
authors.forename: William C
authors.initials: WC
authors.lastname: Sessa
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Iwakiri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051926:Basigin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D015538:Nitrosation / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: C429024:GOLPH3 protein, human; D008565:Membrane Proteins; D020030:Nitric Oxide Donors; D051926:Basigin; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III