Measurement of Protein Glutathionylation

Filipovska, Aleksandra; Murphy, Michael P.

doi:10.1002/0471140856.tx0611s28

Cited by 3 publications

(2 citation statements)

References 16 publications

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“…Protein glutathionylation can be assessed using antibody detection, by HPLC after protein deglutathionylation, and radiolabeled GSH, as well as by mass spectrometry (reviewed in Refs. [91][92][93].…”

Section: Glutathionementioning

confidence: 99%

Reactive Oxygen Species and Oxidative Stress in Obesity—Recent Findings and Empirical Approaches

McMurray

Patten

Harper

2016

Obesity

215

143

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Objective: High levels of reactive oxygen species (ROS) are intricately linked to obesity and associated pathologies, notably insulin resistance and type 2 diabetes. However, ROS are also thought to be important in intracellular signaling, which may paradoxically be required for insulin sensitivity. Many theories have been developed to explain this apparent paradox, which have broadened our understanding of these important small molecules. While many sites for intracellular ROS production have been described, mitochondrial generated ROS remain a major contributor in most cell types. Mitochondrial ROS generation is controlled by a number of factors described in this review. Moreover, these studies have established both a demand for novel sensitive approaches to measure ROS, as well as a need to standardize and review their suitability for different applications. Methods: To properly assess levels of ROS and mitochondrial ROS in the development of obesity and its complications, a growing number of tools have been developed. This paper reviews many of the common methods for the investigation of ROS in mitochondria, cell, animal, and human models. Results: Available approaches can be generally divided into those that measure ROS-induced damage (e.g., DNA, lipid, and protein damage); those that measure antioxidant levels and redox ratios; and those that use novel biosensors and probes for a more direct measure of different forms of ROS (e.g., 2 0 ,7 0 -dichlorofluorescein (DCF), dihydroethidium (DHE) and its mitochondrial targeted form (MitoSOX), Amplex Red, roGFP, HyPer, mt-cpYFP, ratiometric H 2 O 2 probes, and their derivatives). Moreover, this review provides caveats and strengths for the use of these techniques in different models. Conclusions: Advances in these techniques will undoubtedly advance the understanding of ROS in obesity and may help resolve unanswered questions in the field.

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Section: Glutathionementioning

confidence: 99%

Reactive Oxygen Species and Oxidative Stress in Obesity—Recent Findings and Empirical Approaches

McMurray

Patten

Harper

2016

Obesity

215

143

View full text Add to dashboard Cite

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“…The selective reduction of S-glutathionylated cysteine with Grx reduction mixture (GSSG/GR/NADPH), and a specific reaction with the thiol (-SH) group isobaric mass label iodoTMT was used to modify the modified thiol, then resin conjugated with anti-TMT antibodies was used to enrich labeled peptides. The specificity and sensitivity of iodoTMT in labeling the reduced sulfhydryl group, enrichment efficiency of anti-TMT resin, and accuracy and precision of the MS methods have been measured in previous studies [23,48,49]. Combining these results with our quality control data proved our workflow has high specificity, accuracy and precision in the identification and quantification of the modified peptides.…”

Section: Plos Pathogensmentioning

confidence: 62%

S-glutathionylation proteome profiling reveals a crucial role of a thioredoxin-like protein in interspecies competition and cariogenecity of Streptococcus mutans

Zhang

Cheng

et al. 2020

PLoS Pathog

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S-glutathionylation is an important post-translational modification (PTM) process that targets protein cysteine thiols by the addition of glutathione (GSH). This modification can prevent proteolysis caused by the excessive oxidation of protein cysteine residues under oxidative or nitrosative stress conditions. Recent studies have suggested that protein S-glutathionylation plays an essential role in the control of cell-signaling pathways by affecting the protein function in bacteria and even humans. In this study, we investigated the effects of Sglutathionylation on physiological regulation within Streptococcus mutans, the primary etiological agent of human dental caries. To determine the S-glutathionylated proteins in bacteria, the Cys reactive isobaric reagent iodoacetyl Tandem Mass Tag (iodoTMT) was used to label the S-glutathionylated Cys site, and an anti-TMT antibody-conjugated resin was used to enrich the modified peptides. Proteome profiling identified a total of 357 glutathionylated cysteine residues on 239 proteins. Functional enrichment analysis indicated that these Sglutathionylated proteins were involved in diverse important biological processes, such as pyruvate metabolism and glycolysis. Furthermore, we studied a thioredoxin-like protein (Tlp) to explore the effect of S-glutathionylation on interspecies competition between oral streptococcal biofilms. Through site mutagenesis, it was proved that glutathionylation on Cys41 residue of Tlp is crucial to protect S. mutans from oxidative stress and compete with S. sanguinis and S. gordonii. An addition rat caries model showed that the loss of S-glutathionylation attenuated the cariogenicity of S. mutans. Taken together, our study provides an insight into the S-glutathionylation of bacterial proteins and the regulation of oxidative stress resistance and interspecies competition.

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