Abstract
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.
Highlights
Specificity of the Biotin Switch Technique for the Detection of Protein S-Nitrosothiols—To evaluate the specificity of the biotin switch technique (BST), we first sought to perform the assay with an exemplary protein on which we could introduce alternative Cys modifications, including free sulfhydryl (-SH), S-glutathionyl mixed disulfide (-SSG), sulfenic/sulfinic/sulfonic acids (-SOH, -SO2H, -SO3H), and S-nitrosothiol (-SNO)
Biotinylation— the BST appeared to be highly spe- the combination of indirect sunlight and Asc dracific for protein SNOs under our assay conditions, we nonethe- matically increased the biotinylation of SNO-GAPDH, notwithstanding loss of SNO specificity under pendent biotinylation of reduced bovine serum albumin [12]. these conditions. We reasoned that this SNO-independent, Asc-dependent pro- To understand the mechanism of this artifact, we examined tein biotinylation must involve reduction of protein disulfides the ability of Asc to reduce biotin-HPDP to biotin-SH under the or biotin-HPDP
Given the widespread use of the assay, it remains important to show that assay specificity, which has to date been examined for a limited number of proteins [2, 39], captures the general behavior of SNO proteins in complex biological systems
Summary
Shared between these claims of artifactual signals is the idea that Asc must be reducing disulfides (S-glutathionylated or intramolecular disulfides). This notion is difficult to reconcile experimentally with the use of S-methyl methanethiosulfonate as a blocking agent (i.e. protein thiols are S-methylthiolated to form mixed disulfides). The assay would never work if ascorbate removed the blocking agent, and the two eϪ reduction of Cys disulfides to thiol (Cys thiol/disulfide Ered Ϫ170 to Ϫ320 mV) by Asc is highly thermodynamically unfavorable (Asc/DHAsc Ered ϩ70 mV) [13]. The electrochemical measurements favor the reverse reaction (i.e. thiol-dependent reduction of dehydroascorbate (DHAsc0 to Asc), a scenario supported by extensive observations [14, 15]. We sought to employ the BST to differentiate S-nitrosylated from S-oxidized proteins under conditions of diverse oxidative and nitrosative stresses, as such analyses have not been previously possible
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