RGD Reference Report - 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. - Rat Genome Database

RGD Reference Report - 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. - Rat Genome Database

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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.
Authors:	Chouchani, ET Hurd, TR Nadtochiy, SM Brookes, PS Fearnley, IM Lilley, KS Smith, RA Murphy, MP
Citation:	Chouchani ET, etal., Biochem J. 2010 Aug 15;430(1):49-59. doi: 10.1042/BJ20100633.
RGD ID:	7327223
Pubmed:	PMID:20533907 (View Abstract at PubMed)
PMCID:	PMC2911678 (View Article at PubMed Central)
DOI:	DOI:10.1042/BJ20100633 (Journal Full-text)
The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). 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 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. 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 four 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, mitochondrial aldehyde dehydrogenase and alpha-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, whereas 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.

Objects referenced in this article

Gene	Acadvl	acyl-CoA dehydrogenase, very long chain	Rattus norvegicus
Gene	Hspa5	heat shock protein family A (Hsp70) member 5	Rattus norvegicus
Gene	Immt	inner membrane mitochondrial protein	Rattus norvegicus
Gene	Myh6	myosin heavy chain 6	Rattus norvegicus

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