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Isotopic-labelled Clickable Glutathione to Quantify Protein S-Glutathionylation

ABSTRACT: Investigated protein glutathionylation in HL-1 cardiomyocyte cells in response to hydrogen peroxide using a clickable glutathione approach. After exposure, or not, to hydrogen peroxide the glutathionylated proteins with heavy or light azido glutathione were enriched and subjected to LC-MS/MS analysis followed by quantification.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Maheeshi Yapa Abeywardana

LAB HEAD: Young-Hoon Ahn

PROVIDER: PXD014937 | Pride | 2021-09-08

REPOSITORIES: Pride

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			Action	DRS
	2122-Ahn-44A.raw	Raw
	2122-Ahn-44B.raw	Raw
	2122-Ahn-44C.raw	Raw
	2122.txt.dat	Txt
	2122mqpar.xml	Xml

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Publications

Isotopically Labeled Clickable Glutathione to Quantify Protein S-Glutathionylation.

VanHecke Garrett C GC Yapa Abeywardana Maheeshi M Huang Bo B Ahn Young-Hoon YH

Chembiochem : a European journal of chemical biology 20191029 6

Protein S-glutathionylation is one of the important cysteine oxidation events that regulate various redox-mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S-glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido-Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification s ...[more]

PMID: 31560820

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Site-specific Identification of Glutathionylation

Project description:Cardiomyocytes proteins were investigated for their glutathionylation in response to hydrogen peroxide generated from glucose oxidase and using a clickable glutathione derivative. After exposure, or not, to hydrogen peroxide the glutathionylated proteins were enriched and LC-MS/MS analyses was performed.

2019-03-06 | PXD012171 | Pride

Clickable glutathione-based Isotope Coded Affinity Label to quantify glutathionylation

Project description:Identification of cysteines with high oxidation susceptibility is important for understanding redox-mediated biological processes associated with health and disease. We developed a chemical proteomic strategy that helps find cysteines with high susceptibility to S-glutathionylation. Our strategy is based on the isotopically labelled clickable glutathione derivatives that can quantify relative levels of glutathionylated and reduced forms (SSG vs. SH) of cysteines in mass spectrometry, which is named 'Clickable Glutathione-based Isotope-coded Affinity-Tag (G-ICAT).' The G-ICAT approach was applied to the mouse C2C12 cell line upon addition of hydrogen peroxide, finding 1,518 glutathionylated cysteines and their relative levels of SSG over SH upon adding hydron peroxide. This chemical proteomic strategy will significantly contribute to identifying functional cysteines regulated by glutathionylation in redox signaling.

2024-03-07 | PXD034976 | Pride

Clickable Glutathione Approach for Identification of Protein Glutathionylation in Response to Glucose Metabolism

Project description:Glucose metabolism and mitochondrial function are closely interconnected with cellular redox-homeostasis. Although glucose starvation, which often occurs in ischemic heart or insufficient vascularization of tumor cells, is known to induce redox-disturbance, global and individual protein glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. In this report, we use our clickable glutathione approach, which form clickable glutathione (azido-glutathione) by using a mutant of glutathione synthetase (GS M4), for detection and identification of protein glutathionylation in response to glucose starvation. We found that protein glutathionylation is readily induced in response to glucose starvation when mitochondrial reactive oxygen species (ROS) are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Proteomic and biochemical analysis identified over 1,300 proteins, including SMYD2, PP2Cα, and catalase. We further analyzed the cysteine modification site and functional implication of PP2Cα glutathionylation.

2018-10-26 | PXD004026 | Pride

Site-specific Identification and Quantification of Protein Glutathionylation under Ischemic Stress

Project description:Ischemia or ischemic reperfusion is well-known for its contribution to heart diseases. During ischemia and reperfusion, a flux of nutrients and oxygen to cardiomyocytes is altered, which causes a burst of ROS from mitochondria and other enzymes. Elevated levels of ROS can cause oxidative modifications of cardiac proteins, such as protein glutathionylation. Despite previous extensive studies, proteomic identification of glutathionylated proteins in cardiomyocytes under altered levels of nutrients and oxygen has been relatively limited. We have applied our clickable glutathione approach to HL-1 cardiomyocyte cell line under metabolic alterations of glucose, oxygen, and fatty acids to detect and identify proteins undergoing glutathionylation.

2021-08-13 | PXD024766 | Pride

In-vitro glutathionylation of SMYD2

Project description:Glutathionylation is an important post-translational modification and developing tools to identify which cysteine residues on proteins are glutathionylated in response to different physiological conditions is necessary. This project utilizes an approach consisting of azido-glutathione, a single purified protein, and an oxidant to form disulfide bonds between the azide modified glutathione and the cysteine residues of the protein. After this the reaction is quenched with iodoacetamide, subjected to a click reaction with a chemically cleavable alkyne conjugated biotin, and digested with trypsin. These peptide fragments are eluted from streptavidin beads and subjected to LC-MS/MS to determine which cysteine residues are glutathionylated in-vitro.

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Transcription profiling of Saccharomyces cerevisiae reveals the Cbf1 basic helix-loop-helix transcription factor is required for the response to hydrogen peroxide stress

Project description:Cbf1 is a basic helix-loop-helix transcription factor which regulates the expression of many genes of the sulphur assimilation pathway in yeast. Gamma-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis, is constitutively elevated in cbf1 mutants indicating that Cbf1 normally represses GSH1 expression. We used transcriptional profiling to show that a number of antioxidant and stress genes are similarly repressed by Cbf1, consistent with the high oxidant tolerance of cbf1 mutants. Our data indicate that Cbf1 plays a key role in the regulation of gene expression during oxidative stress conditions induced by exposure to hydrogen peroxide. The Yap1 transcription factor induces GSH1 expression in response to hydrogen peroxide in a mechanism that does not require the Met4 transcription factor to overcome the inhibitory action of Cbf1. We show that hydrogen peroxide does not affect Cbf1 occupancy on the GSH1 promoter, but results in specific modification of Cbf1 by phosphorylation. Our data indicate that casein kinase (CK2) can directly phosphorylate Cbf1 in vitro. Furthermore, CK2 activity is required to phosphorylate Cbf1 and induce GSH1 expression in yeast cells in response to hydrogen peroxide. CK2 mutants are sensitive to hydrogen peroxide consistent with a role for CK2 in regulating the cellular response to oxidative stress. CK2 is a ubiquitous serine/threonine protein kinase and the finding that it is regulated by oxidative stress is particularly interesting, since there is increasing evidence that it is involved in a number of pathological conditions including cancer, neurodegenerative and cardiovascular diseases.

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Expression of Heterologous OsDHAR Gene Improves Glutathione (GSH)-Dependent Antioxidant System and Maintenance of Cellular Redox Status in Synechococcus elongatus PCC 7942

Project description:An excess of reactive oxygen species (ROS) can cause severe oxidative damage to cellular components in photosynthetic cells. Antioxidant systems, such as the ascorbate-glutathione cycle, regulate redox status in cells to guard against such damage. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of oxidized ascorbate (dehydroascorbate) and contains a redox active site and glutathione binding-site. The DHAR gene is important in biological and abiotic stress responses involving reduction of the oxidative damage caused by ROS. In this study, a transgenic microalgae (TA) strain was constructed by cloning the Oryza sativa L. japonica DHAR (OsDHAR) gene controlled by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter (Ptrc) into the Synechococcus elongatus PCC 7942 strain of cyanobacteria to study the functional activities of OsDHAR under oxidative stress caused by hydrogen peroxide exposure. OsDHAR expression increased the growth of S. elongatus PCC 7942 under oxidative stress by reducing the levels of hydroperoxides and malondialdehyde (MDA) and mitigating the loss of chlorophyll. DHAR and glutathione S-transferase activity were higher than in the wild-type (WT) strain. Additionally, overexpression of OsDHAR in S. elongatus PCC 7942 greatly increased the glutathione (GSH)/glutathione disulfide (GSSG) ratio compared to ascorbic acid (AsA)/dehydroascorbate (DHA) ratio in the presence or absence of hydrogen peroxide. These results strongly suggest that DHAR attenuates deleterious oxidative effects via the glutathione (GSH)-dependent antioxidant system in cyanobacterial cells. The expression of heterologous OsDHAR in S. elongatus PCC 7942 protected cells from oxidative damage through a GSH-dependent antioxidant system via GSH-dependent reactions at the redox active site and GSH binding site residues during oxidative stress.

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Hydrogen peroxide treatment of Jurkat T lymphoma cells leads to upregulation of genes involved in intracellular signalling pathways

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Project description:Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes multiple infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and acquired drug-resistance mechanisms, A. baumannii isolates are commonly multi-drug resistant and infections are notoriously difficult to treat. Therefore, it is important to identify mechanisms used by A. baumannii to survive stresses encountered during infection as a means of identifying new drug targets. In this study, we determined the transcriptional response of A. baumannii to hydrogen peroxide stress using RNASequencing. Upon exposure to hydrogen peroxide, A. baumannii differentially transcribes several hundred genes. In this study, we also determined the transcriptional profile of A. baumannii strains with the transcriptional regulators mumR or oxyR genetically inactivated and identified transcriptional differences between these strains and wild-type A. baumannii in response to hydrogen peroxide stress. In doing this, the function of A. baumannii OxyR in hydrogen peroxide stress resistance and regulation of genes required for hydrogen peroxide detoxification was defined. Moreover, the contribution of the uncharacterized regulator MumR to hydrogen peroxide stress resistance was also explored. This work reveals the transcriptome of an important human pathogen in the presence of hydrogen peroxide stress.

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