Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol Difference In Gel Electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochemical Journal Année : 2010

Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol Difference In Gel Electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation

Edward Chouchani
  • Fonction : Auteur
Thomas Hurd
  • Fonction : Auteur
Sergiy M Nadtochiy
  • Fonction : Auteur
Paul S Brookes
  • Fonction : Auteur
Ian M. Fearnley
  • Fonction : Auteur
Kathryn S Lilley
  • Fonction : Auteur
Robin Andrew James Smith
  • Fonction : Auteur

Résumé

The S-nitrosation of mitochondrial proteins as a consequence of nitric oxide (NO) metabolism is of physiological and pathological significance. We previously developed a mitochondria-targeted S-nitrosothiol (MitoSNO) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, S-nitrosothiol Difference In Gel Electrophoresis (SNO-DIGE). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 or Cy5 fluorescent tags, mixed together and individual protein spots were resolved by 2D gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by mass spectrometry. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further 4 that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, aldehyde dehydrogenase and a-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, while identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.

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hal-00506525 , version 1 (28-07-2010)

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Edward Chouchani, Thomas Hurd, Sergiy M Nadtochiy, Paul S Brookes, Ian M. Fearnley, et al.. Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol Difference In Gel Electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation. Biochemical Journal, 2010, 430 (1), pp.49-59. ⟨10.1042/BJ20100633⟩. ⟨hal-00506525⟩

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