Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress.
Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action.
Forrester
Michael T
MT
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Foster
Matthew W
MW
Stamler
Jonathan S
JS
D001205:Ascorbic Acid / Q000494:pharmacology; D001710:Biotin / Q000378:metabolism*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D003545:Cysteine / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010782:Photolysis; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013472:Sunlight
D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D001710:Biotin; D019001:Nitric Oxide Synthase; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D005978:Glutathione; D003545:Cysteine; D009584:Nitrogen; D001205:Ascorbic Acid; D019331:NG-Nitroarginine Methyl Ester