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Jonathan S Stamler - One of the best experts on this subject based on the ideXlab platform.
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A Multiplex Enzymatic Machinery for Cellular Protein S-Nitrosylation
Molecular Cell, 2018Co-Authors: Divya Seth, Douglas T. Hess, Alfred Hausladen, Yajuan Wang, Liwen Wang, Jonathan S StamlerAbstract:Summary S-Nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-Nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-Nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-Nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-Nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling.
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protein s Nitrosylation determinants of specificity and enzymatic regulation of s nitrosothiol based signaling
Antioxidants & Redox Signaling, 2018Co-Authors: Colin T. Stomberski, Jonathan S StamlerAbstract:Abstract Significance: Protein S-Nitrosylation, the oxidative modification of cysteine by nitric oxide (NO) to form protein S-nitrosothiols (SNOs), mediates redox-based signaling that conveys, in large part, the ubiquitous influence of NO on cellular function. S-Nitrosylation regulates protein activity, stability, localization, and protein–protein interactions across myriad physiological processes, and aberrant S-Nitrosylation is associated with diverse pathophysiologies. Recent Advances: It is recently recognized that S-Nitrosylation endows S-nitroso-protein (SNO-proteins) with S-nitrosylase activity, that is, the potential to trans-S-nitrosylate additional proteins, thereby propagating SNO-based signals, analogous to kinase-mediated signaling cascades. In addition, it is increasingly appreciated that cellular S-Nitrosylation is governed by dynamically coupled equilibria between SNO-proteins and low-molecular-weight SNOs, which are controlled by a growing set of enzymatic denitrosylases comprising two ma...
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Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1.
PLOS ONE, 2016Co-Authors: Divya Seth, Jonathan S Stamler, Fabio V. Fonseca, Liwen Wang, Tsan Sam Xiao, Phillip Gruber, Alan M. TartakoffAbstract:Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-Nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-Nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-Nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-Nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-Nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-Nitrosylation affects the biology of this protein in vivo.
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s Nitrosylation of the mitochondrial chaperone trap1 sensitizes hepatocellular carcinoma cells to inhibitors of succinate dehydrogenase
Cancer Research, 2016Co-Authors: Salvatore Rizza, Simone Cardaci, Costanza Montagna, Giuseppina Di Giacomo, Daniela De Zio, Emiliano Maiani, Virginia Sanchezquiles, Blagoy Blagoev, Andrea Rasola, Jonathan S StamlerAbstract:S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-Nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-Nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-Nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-Nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S-Nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 4170-82. ©2016 AACR.
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Identification of S-nitroso-CoA reductases that regulate protein S-Nitrosylation.
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Puneet Anand, Douglas T. Hess, Alfred Hausladen, Yajuan Wang, Guofang Zhang, Colin T. Stomberski, Henri Brunengraber, Jonathan S StamlerAbstract:Coenzyme A (CoA) mediates thiol-based acyl-group transfer (acetylation and palmitoylation). However, a role for CoA in the thiol-based transfer of NO groups (S-Nitrosylation) has not been considered. Here we describe protein S-Nitrosylation in yeast (heretofore unknown) that is mediated by S-nitroso-CoA (SNO-CoA). We identify a specific SNO-CoA reductase encoded by the alcoholdehydrogenase 6 (ADH6) gene and show that deletion of ADH6 increases cellular S-Nitrosylation and alters CoA metabolism. Further, we report that Adh6, acting as a selective SNO-CoA reductase, protects acetoacetyl–CoA thiolase from inhibitory S-Nitrosylation and thereby affects sterol biosynthesis. Thus, Adh6-regulated, SNO-CoA–mediated protein S-Nitrosylation provides a regulatory mechanism paralleling protein acetylation. We also find that SNO-CoA reductases are present from bacteria to mammals, and we identify aldo-keto reductase 1A1 as the mammalian functional analog of Adh6. Our studies reveal a novel functional class of enzymes that regulate protein S-Nitrosylation from yeast to mammals and suggest that SNO-CoA–mediated S-Nitrosylation may subserve metabolic regulation.
A. Riccio - One of the best experts on this subject based on the ideXlab platform.
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s Nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons
Nature, 2008Co-Authors: Alexi Nott, Marc P Watson, James D Robinson, Luca Crepaldi, A. RiccioAbstract:The neurotrophin BDNF (brain-derived neurotrophic factor) influences the expression of many genes in the developing nervous system and is known to induce nitric oxide. Nott et al. show that BDNF triggers nitric oxide synthesis and accompanying S-Nitrosylation of several proteins in the nucleus of cortical neurons, including the histone deacetylase HDAC2. Nitrosylation of HDAC2 leads to its release from the chromatin of neurotrophin-dependent genes, which allows histone acetylation and active transcription. Brain-derived neurotrophic factor (BDNF) and other neurotrophins have a vital role in the development of the rat and mouse nervous system by influencing the expression of many specific genes that promote differentiation, cell survival, synapse formation and, later, synaptic plasticity1. Although nitric oxide (NO) is known to be an important mediator of BDNF signalling in neurons2, the mechanisms by which neurotrophins influence gene expression during development and plasticity remain largely unknown. Here we show that BDNF triggers NO synthesis and S-Nitrosylation of histone deacetylase 2 (HDAC2) in neurons, resulting in changes to histone modifications and gene activation. S-Nitrosylation of HDAC2 occurs at Cys 262 and Cys 274 and does not affect deacetylase activity. In contrast, Nitrosylation of HDAC2 induces its release from chromatin, which increases acetylation of histones surrounding neurotrophin-dependent gene promoters and promotes transcription. Notably, Nitrosylation of HDAC2 in embryonic cortical neurons regulates dendritic growth and branching, possibly by the activation of CREB (cyclic-AMP-responsive-element-binding protein)-dependent genes. Thus, by stimulating NO production and S-Nitrosylation of HDAC2, neurotrophic factors promote chromatin remodelling and the activation of genes that are associated with neuronal development.
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S-Nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons
NATURE, 2008Co-Authors: A. RiccioAbstract:Brain- derived neurotrophic factor ( BDNF) and other neurotrophins have a vital role in the development of the rat and mouse nervous system by influencing the expression of many specific genes that promote differentiation, cell survival, synapse formation and, later, synaptic plasticity(1). Although nitric oxide ( NO) is known to be an important mediator of BDNF signalling in neurons(2), the mechanisms by which neurotrophins influence gene expression during development and plasticity remain largely unknown. Here we show that BDNF triggers NO synthesis and S- Nitrosylation of histone deacetylase 2 ( HDAC2) in neurons, resulting in changes to histone modifications and gene activation. S- Nitrosylation of HDAC2 occurs at Cys 262 and Cys 274 and does not affect deacetylase activity. In contrast, Nitrosylation of HDAC2 induces its release from chromatin, which increases acetylation of histones surrounding neurotrophin- dependent gene promoters and promotes transcription. Notably, Nitrosylation of HDAC2 in embryonic cortical neurons regulates dendritic growth and branching, possibly by the activation of CREB ( cyclic- AMP-responsive-element- binding protein)- dependent genes. Thus, by stimulating NO production and S- Nitrosylation of HDAC2, neurotrophic factors promote chromatin remodelling and the activation of genes that are associated with neuronal development.
Yasuko Iwakiri - One of the best experts on this subject based on the ideXlab platform.
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proteomic identification of s nitrosylated golgi proteins new insights into endothelial cell regulation by enos derived no
PLOS ONE, 2012Co-Authors: Panjamaporn Sangwung, Todd M Greco, William C Sessa, Yanzhuang Wang, Yasuko IwakiriAbstract: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.
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Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO
2012Co-Authors: Panjamaporn Sangwung, William C Sessa, Todd M Greco, Harry Ischiropoulos, Yanzhuang Wang, Yasuko IwakiriAbstract:BackgroundEndothelial 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. MethodsGolgi 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. ResultsSeventy-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. ConclusionOur 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.
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nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein s Nitrosylation and protein trafficking
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Yasuko Iwakiri, Ayano Satoh, Suvro Chatterjee, Derek Toomre, Cecile Chalouni, David Fulton, Roberto J Groszmann, Vijay H Shah, William C SessaAbstract:Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-Nitrosylation. Although intracellular S-Nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-Nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-Nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-Nitrosylation, the specific S-Nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-Nitrosylation reactions that can regulate intracellular transport processes.
Todd M Greco - One of the best experts on this subject based on the ideXlab platform.
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Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS- Derived NO
2016Co-Authors: Panjamaporn Sangwung, Todd M Greco, Harry Ischiropoulos, Yanzhuang Wang, William C, Sessa Yasuko IwakiriAbstract: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
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proteomic identification of s nitrosylated golgi proteins new insights into endothelial cell regulation by enos derived no
PLOS ONE, 2012Co-Authors: Panjamaporn Sangwung, Todd M Greco, William C Sessa, Yanzhuang Wang, Yasuko IwakiriAbstract: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.
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Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO
2012Co-Authors: Panjamaporn Sangwung, William C Sessa, Todd M Greco, Harry Ischiropoulos, Yanzhuang Wang, Yasuko IwakiriAbstract:BackgroundEndothelial 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. MethodsGolgi 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. ResultsSeventy-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. ConclusionOur 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.
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structural profiling of endogenous s nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein s Nitrosylation
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Paschalis-Thomas Doulias, Margarita Tenopoulou, Jennifer L Greene, Todd M Greco, Steve H Seeholzer, Roland L DunbrackAbstract:S-Nitrosylation, the selective posttranslational modification of protein cysteine residues to form S-nitrosocysteine, is one of the molecular mechanisms by which nitric oxide influences diverse biological functions. In this study, unique MS-based proteomic approaches precisely pinpointed the site of S-Nitrosylation in 328 peptides in 192 proteins endogenously modified in WT mouse liver. Structural analyses revealed that S-nitrosylated cysteine residues were equally distributed in hydrophobic and hydrophilic areas of proteins with an average predicted pK(a) of 10.01 ± 2.1. S-Nitrosylation sites were over-represented in α-helices and under-represented in coils as compared with unmodified cysteine residues in the same proteins (χ(2) test, P < 0.02). A quantile-quantile probability plot indicated that the distribution of S-nitrosocysteine residues was skewed toward larger surface accessible areas compared with the unmodified cysteine residues in the same proteins. Seventy percent of the S-nitrosylated cysteine residues were surrounded by negatively or positively charged amino acids within a 6-A distance. The location of cysteine residues in α-helices and coils in highly accessible surfaces bordered by charged amino acids implies site directed S-Nitrosylation mediated by protein-protein or small molecule interactions. Moreover, 13 modified cysteine residues were coordinated with metals and 15 metalloproteins were endogenously modified supporting metal-catalyzed S-Nitrosylation mechanisms. Collectively, the endogenous S-nitrosoproteome in the liver has structural features that accommodate multiple mechanisms for selective site-directed S-Nitrosylation.
William C Sessa - One of the best experts on this subject based on the ideXlab platform.
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proteomic identification of s nitrosylated golgi proteins new insights into endothelial cell regulation by enos derived no
PLOS ONE, 2012Co-Authors: Panjamaporn Sangwung, Todd M Greco, William C Sessa, Yanzhuang Wang, Yasuko IwakiriAbstract: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.
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Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO
2012Co-Authors: Panjamaporn Sangwung, William C Sessa, Todd M Greco, Harry Ischiropoulos, Yanzhuang Wang, Yasuko IwakiriAbstract:BackgroundEndothelial 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. MethodsGolgi 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. ResultsSeventy-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. ConclusionOur 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.
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nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein s Nitrosylation and protein trafficking
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Yasuko Iwakiri, Ayano Satoh, Suvro Chatterjee, Derek Toomre, Cecile Chalouni, David Fulton, Roberto J Groszmann, Vijay H Shah, William C SessaAbstract:Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-Nitrosylation. Although intracellular S-Nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-Nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-Nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-Nitrosylation, the specific S-Nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-Nitrosylation reactions that can regulate intracellular transport processes.