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Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. We have investigated modifications to protein thiols in enriched mitochondrial fractions using a modified form of 2D electrophoresis that we have called Redox-DIGE. We have observed a number of cysteine residues that are sensitive enough to respond to plausible in vivo concentrations (low µM) of reactive oxygen or nitrogen species [1] [2].

We have used a redox proteomic technique called oxidative isotope-coded affinity tags (OxICAT) to assess cysteine-residue redox changes. We used this approach to simultaneously identify and quantify the redox state of several hundred cysteine residues in Drosophila melanogaster in vivo during ageing and fasting [3]. We have also used it to show that in mouse in vivo protein S-nitrosation occurs in response to the combination of ischemia and nitrite [4].

Current work in the lab has focused on non-enzymatic reversible cysteine S-acetylation/S-acylation by acetyl-CoA and how this impacts on irreversible lysine N-acetylation/N-acylation. Surface cysteine residues readily react with acyl-CoAs and once the acyl moiety is localised on the surface of the protein it can be transferred to nearby lysine residues in a reaction that is enhanced by proximity [5][6]. These reactive sites on the surfaces of protein are less conserved within the genomes of vertebrates [6]. Current work in the lab is focused on why this low stoichiometry (~0.1%) acylation is detrimental.



  1. Hurd TR, Prime TA, Harbour ME, Lilley KS & Murphy MP (2007)
    Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling.
    J Biol Chem 282, 22040-51
  2. Chouchani ET, Hurd TR, Nadtochiy SM, Brookes PS, Fearnley IM, Lilley KS, Smith RAJ & Murphy MP (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.
    Biochem J 430, 49-59
  3. Menger KE, James AM, Cochemé HM, Harbour ME, Chouchani ET, Ding S, Fearnley IM, Partridge L & Murphy MP (2015)
    Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster.
    Cell Rep 11, 1856-65
  4. Chouchani ET, James AM, Methner C, Pell VR, Prime TA, Erikson BK, Forkink M, Lau GY, Bright TP, Menger KE, Fearnley IM, Krieg T & Murphy MP (2017)
    Identification and Quantification of Protein S-nitrosation by Nitrite in the Mouse Heart during Ischemia.
    J Biol Chem 35, 1486-14495
  5. James AM, Hoogewijs K, Logan A, Hall AR, Ding S, Fearnley IM & Murphy MP (2017)
    Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II.
    Cell Rep 18, 2105-2112
  6. James AM, Smith AC, Smith CL, Robinson AJ & Murphy MP (2018)
    Proximal Cysteines that Enhance Lysine N-Acetylation of Cytosolic Proteins in Mice Are Less Conserved in Longer-Living Species.
    Cell Rep 24, 1445-1455