Development of ‘Redox Arrays’ for identifying novel glutathionylated proteins in the secretome

Abstract

Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques.

Store-operated Ca2+ entry controls ameloblast cell function and enamel development

“…Glutathione and S-transferases are also important for the S-glutathionylation of a number of proteins with cysteine residues (35,36). Two common target proteins of S-glutathionylation are keratins and actin (22,37). EO cells from Stim1/2 K14cre mice showed a substantial downregulation of keratins and decreased development of tonofibrils, consistent with altered glutathione system.…”

“…Changes in the glutathione system had a broader effect. For example, keratins, which serve as a part of the ameloblast's cytoskeleton forming tonofilaments (tonofibrils) (21), are known to undergo S-glutathionylation (22), which can have an effect in ameloblast morphology. RNAseq data showed that mRNA expression of many keratin genes was significantly downregulated in Stim1/2-deficient ameloblasts, including Krt4, Krt13, Krt16, Krt19, and Krt27 (Supplemental Figure 4, A and B).…”

Positive Regulation of Interleukin-1β Bioactivity by Physiological ROS-Mediated Cysteine S-Glutathionylation

“…Large-scale proteomic studies have identified numerous redox-sensitive proteins including some extracellular proteins. For instance, two recent elegant studies identified a set of glutathionylated proteins released during inflammation (Checconi et al, 2015; Mullen et al, 2015). Nevertheless, likely due to the extremely low levels of production, none of the cytokines (including IL-1β) were identified as glutathionylation targets in the previous studies.…”

Oxidative Stress and Inflammation Induced by Environmental and Psychological Stressors: A Biomarker Perspective

“…Pre-peer-review manuscript 27 In the context of oxidative stress, several redox proteomics techniques and their application to the field of inflammation and neuroinflammation have led to the identification of specific oxidized proteins undergoing carbonylation (149) or glutathionylation (26,103). The extension of omics to oxidative post-translational modifications may add a dimension to the information obtained and eventually provide a more precise way of identifying exposure to agents that cause oxidative stress.…”