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Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

ABSTRACT: 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.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.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.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.

SUBMITTER: Sangwung P

PROVIDER: S-EPMC3283662 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

Sangwung Panjamaporn P Greco Todd M TM Wang Yanzhuang Y Ischiropoulos Harry H Sessa William C WC Iwakiri Yasuko Y

PloS one 20120221 2

<h4>Background</h4>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 ce ...[more]

PMID: 22363674

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Project description:Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether S-nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 ?M S-nitrosoglutathione (GSNO) treatment was found to have a maximum biological effect on promoting adventitious rooting in cucumber. Meanwhile, removal of endogenous NO significantly inhibited the development of adventitious roots implying that NO is responsible for promoting the process of adventitious rooting. Moreover, application of GSNO resulted in an increase of intracellular S-nitrosothiol (SNO) levels and endogenous NO production, while decreasing the S-nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that S-nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of S-nitrosylated proteins was performed utilizing the liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and biotin-switch technique during the development of adventitious rooting. Among these proteins, the activities and S-nitrosylated level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin alpha chain (TUA), and glutathione reductase (GR) were further analyzed as NO direct targets. Our results indicated that NO might enhance the S-nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, S-nitrosylation of TUA increased the expression level of TUA. The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting. Collectively, these results suggest that S-nitrosylation could be involved in NO-induced adventitious rooting, and they also provide fundamental evidence for the molecular mechanism of NO signaling during adventitious rooting in cucumber explants.

Dataset Information

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

Publications

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

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