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Proteomic Identification of Protein Glutathionylation in Cardiomyocytes.

ABSTRACT: Reactive oxygen species (ROS) are important signaling molecules, but their overproduction is associated with many cardiovascular diseases, including cardiomyopathy. ROS induce various oxidative modifications, among which glutathionylation is one of the significant protein oxidations that occur under oxidative stress. Despite previous efforts, direct and site-specific identification of glutathionylated proteins in cardiomyocytes has been limited. In this report, we used a clickable glutathione approach in a HL-1 mouse cardiomyocyte cell line under exposure to hydrogen peroxide, finding 1763 glutathionylated peptides with specific Cys modification sites, which include many muscle-specific proteins. Bioinformatic and cluster analyses found 125 glutathionylated proteins, whose mutations or dysfunctions are associated with cardiomyopathy, many of which include sarcomeric structural and contractile proteins, chaperone, and other signaling or regulatory proteins. We further provide functional implication of glutathionylation for several identified proteins, including CSRP3/MLP and complex I, II, and III, by analyzing glutathionylated sites in their structures. Our report establishes a chemoselective method for direct identification of glutathionylated proteins and provides potential target proteins whose glutathionylation may contribute to muscle diseases.

SUBMITTER: VanHecke GC

PROVIDER: S-EPMC6512321 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Proteomic Identification of Protein Glutathionylation in Cardiomyocytes.

VanHecke Garrett C GC Abeywardana Maheeshi Yapa MY Ahn Young-Hoon YH

Journal of proteome research 20190311 4

Reactive oxygen species (ROS) are important signaling molecules, but their overproduction is associated with many cardiovascular diseases, including cardiomyopathy. ROS induce various oxidative modifications, among which glutathionylation is one of the significant protein oxidations that occur under oxidative stress. Despite previous efforts, direct and site-specific identification of glutathionylated proteins in cardiomyocytes has been limited. In this report, we used a clickable glutathione ap ...[more]

PMID: 30831029

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Project description:S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols, and covalent capture of reduced thiols using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was initially validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG modification compared to controls. This approach was extended to identify potential SSG-modified Cys sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible to S-glutathionylation under stress conditions.

Project description:AimsProtein S-glutathionylation is a widely distributed post-translational modification of thiol groups with glutathione that can function as a redox-sensitive switch to mediate redox regulation and signal transduction. The malaria parasite Plasmodium falciparum is exposed to intense oxidative stress and possesses the enzymatic system required to regulate protein S-glutathionylation, but despite its potential importance, protein S-glutathionylation has not yet been studied in malaria parasites. In this work we applied a method based on enzymatic deglutathionylation, affinity purification of biotin-maleimide-tagged proteins, and proteomic analyses to characterize the Plasmodium glutathionylome.ResultsWe identified 493 targets of protein S-glutathionylation in Plasmodium. Functional profiles revealed that the targets are components of central metabolic pathways, such as nitrogen compound metabolism and protein metabolism. Fifteen identified proteins with important functions in metabolic pathways (thioredoxin reductase, thioredoxin, thioredoxin peroxidase 1, glutathione reductase, glutathione S-transferase, plasmoredoxin, mitochondrial dihydrolipoamide dehydrogenase, glutamate dehydrogenase 1, glyoxalase I and II, ornithine ?-aminotransferase, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase [PK], and phosphoglycerate mutase) were further analyzed to study their ability to form mixed disulfides with glutathione. We demonstrate that P. falciparum GAPDH, PK, and ornithine ?-aminotransferase are reversibly inhibited by S-glutathionylation. Further, we provide evidence that not only P. falciparum glutaredoxin 1, but also thioredoxin 1 and plasmoredoxin are able to efficiently catalyze protein deglutathionylation.InnovationWe used an affinity-purification based proteomic approach to characterize the Plasmodium glutathionylome.ConclusionOur results indicate a wide regulative use of S-glutathionylation in the malaria parasite and contribute to our understanding of redox-regulatory processes in this pathogen.

Project description:Leber's hereditary optic neuropathy (LHON, MIM#535000) is the most common form of inherited optic neuropathies and mitochondrial DNA-related diseases. The pathogenicity of mutations in genes encoding components of mitochondrial Complex I is well established, but the underlying pathomechanisms of the disease are still unclear. Hypothesizing that oxidative stress related to Complex I deficiency may increase protein S-glutathionylation, we investigated the proteome-wide S-glutathionylation profiles in LHON (n = 11) and control (n = 7) fibroblasts, using the GluICAT platform that we recently developed. Glutathionylation was also studied in healthy fibroblasts (n = 6) after experimental Complex I inhibition. The significantly increased reactive oxygen species (ROS) production in the LHON group by Complex I was shown experimentally. Among the 540 proteins which were globally identified as glutathionylated, 79 showed a significantly increased glutathionylation (p < 0.05) in LHON and 94 in Complex I-inhibited fibroblasts. Approximately 42% (33/79) of the altered proteins were shared by the two groups, suggesting that Complex I deficiency was the main cause of increased glutathionylation. Among the 79 affected proteins in LHON fibroblasts, 23% (18/79) were involved in energetic metabolism, 31% (24/79) exhibited catalytic activity, 73% (58/79) showed various non-mitochondrial localizations, and 38% (30/79) affected the cell protein quality control. Integrated proteo-metabolomic analysis using our previous metabolomic study of LHON fibroblasts also revealed similar alterations of protein metabolism and, in particular, of aminoacyl-tRNA synthetases. S-glutathionylation is mainly known to be responsible for protein loss of function, and molecular dynamics simulations and 3D structure predictions confirmed such deleterious impacts on adenine nucleotide translocator 2 (ANT2), by weakening its affinity to ATP/ADP. Our study reveals a broad impact throughout the cell of Complex I-related LHON pathogenesis, involving a generalized protein stress response, and provides a therapeutic rationale for targeting S-glutathionylation by antioxidative strategies.

Dataset Information

Proteomic Identification of Protein Glutathionylation in Cardiomyocytes.

Publications

Proteomic Identification of Protein Glutathionylation in Cardiomyocytes.

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