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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

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, her...

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Autores principales: Chouchani, Edward T., Hurd, Thomas R., Nadtochiy, Sergiy M., Brookes, Paul S., Fearnley, Ian M., Lilley, Kathryn S., Smith, Robin A. J., Murphy, Michael P.
Formato: Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911678/
https://www.ncbi.nlm.nih.gov/pubmed/20533907
http://dx.doi.org/10.1042/BJ20100633
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author Chouchani, Edward T.
Hurd, Thomas R.
Nadtochiy, Sergiy M.
Brookes, Paul S.
Fearnley, Ian M.
Lilley, Kathryn S.
Smith, Robin A. J.
Murphy, Michael P.
author_facet Chouchani, Edward T.
Hurd, Thomas R.
Nadtochiy, Sergiy M.
Brookes, Paul S.
Fearnley, Ian M.
Lilley, Kathryn S.
Smith, Robin A. J.
Murphy, Michael P.
author_sort Chouchani, Edward T.
collection PubMed
description 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 α-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.
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spelling pubmed-29116782010-08-02 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 Chouchani, Edward T. Hurd, Thomas R. Nadtochiy, Sergiy M. Brookes, Paul S. Fearnley, Ian M. Lilley, Kathryn S. Smith, Robin A. J. Murphy, Michael P. Biochem J Research Article 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 α-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. Portland Press Ltd. 2010-07-28 2010-08-15 /pmc/articles/PMC2911678/ /pubmed/20533907 http://dx.doi.org/10.1042/BJ20100633 Text en © 2010 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by-nc/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Chouchani, Edward T.
Hurd, Thomas R.
Nadtochiy, Sergiy M.
Brookes, Paul S.
Fearnley, Ian M.
Lilley, Kathryn S.
Smith, Robin A. J.
Murphy, Michael P.
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
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911678/
https://www.ncbi.nlm.nih.gov/pubmed/20533907
http://dx.doi.org/10.1042/BJ20100633
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