Redox proteomics optimized protocol applyed to melanoma cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap XL ETD
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Malignant Cell
SUBMITTER: 
Michel Batista
LAB HEAD: Glaucia Regina Martinez
PROVIDER: PXD049252 | Pride | 2024-02-28
REPOSITORIES: Pride
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Project description:Abstract of associated menuscript; Quinones and alpha,beta-unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol-(S)-alkylation. We analyzed the global expression profile of Bacillus subtilis to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol-reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol-dependent formaldehyde dehydrogenase (AdhA) and DJ-1/PfpI-family cysteine proteinase (YraA). The adhA-yraA operon and the yraC gene, encoding gamma-carboxymuconolactone decarboxylase, are positively regulated by the MerR-family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7-4-7 inverted repeat (CTTAAAG-N4-CTTTAAG) between the -35 and -10 elements. Activation of adhA-yraA transcription by AdhR requires the conserved Cys52 residue in vivo. We speculate that AdhR is redox-regulated via thiol-(S)-alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol-containing proteins, respectively. WT (-FA) vs. WT (+ 1 mM FA), WT (-MG) vs. WT (+ 2.8 mM MG), WT (-MG) vs. WT (+ 5.6 mM MG). Each experiement was conducted triplicates using three independent total RNA preparations. For all datasets used for final analysis, untreated samples were labeled with Cy3 and treated samples with Cy5.
2010-05-15 | E-GEOD-13925 | biostudies-arrayexpress
Project description:Thioredoxin/glutathione reductase (TGR, TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains; the function of this enzyme remains unknown. This NADPH-dependent thiol oxidoreductase evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.
2022-10-13 | PXD033872 | Pride
Project description:This proteomic study This proteomic study focuses on the role of reversible thiol oxidation after hexyl aminolevulinate mediated PDT (HAL-PDT). The human epidermoid carcinoma cell line A431 was used as model system and red light as light source, a clinical relevant in vitro model. The light dose dependent cell cytotoxicity was measured by resazurin assay. The most clinical relevant dose, LD100, was used for the proteomic part of the study and cells where harvested 30 min after treatment. The reversibly oxidized thiol proteins were selected by biotinylation and identified by mass spectrometry. This proteomic technique revealed over 1100 thiol proteins which were reversibly oxidized after HAL-PDT. Identified proteins were analysed by bioinformatics (IPA and GO) and a list of reliable identified proteins (282 proteins) where further analysed. Both the cellular location and function was determined for these proteins. In addition, 18 of the proteins where identified as redox regulated, 36 to be a part of the apoptotic pathway and 9 to be both redox regulated and a part of apoptotic pathway. From these results we suggest redox regulated proteins as a trigger mechanism for PDT induced apoptosis.focuses on the role of reversible thiol oxidation after hexyl aminolevulinate mediated PDT (HAL-PDT). The human epidermoid carcinoma cell line A431 was used as model system and red light as light source, a clinical relevant in vitro model. The light dose dependent cell cytotoxicity was measured by resazurin assay. The most clinical relevant dose, LD100, was used for the proteomic part of the study and cells where harvested 30 min after treatment. The reversibly oxidized thiol proteins were selected by biotinylation and identified by mass spectrometry. This proteomic technique revealed over 1100 thiol proteins which were reversibly oxidized after HAL-PDT. Identified proteins were analysed by bioinformatics (IPA and GO) and a list of reliable identified proteins (282 proteins) where further analysed. Both the cellular location and function was determined for these proteins. In addition, 18 of the proteins where identified as redox regulated, 36 to be a part of the apoptotic pathway and 9 to be both redox regulated and a part of apoptotic pathway. From these results we suggest redox regulated proteins as a trigger mechanism for PDT induced apoptosis.
2016-01-12 | PXD001582 | Pride
Project description:Thiol-based redox regulation is a crucial post-translational mechanism to acclimate plants to changing light availability. Here, we conduct a biotin-switch-based redox proteomics study to systematically investigate dynamics of the thiol-redox network in response to temporal changes in light availability and across genotypes lacking parts of the NTRC/thioredoxin (Trx) systems in the chloroplast. Temporal dynamics revealed light leading to marked decreases in the oxidation states of 75 chloroplast proteins mainly involved in photosynthesis during the first 10 min, followed by their partial re-oxidation after 2-6 hours into the photoperiod. This involved f, m and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys-Prx and Trx y2 showed an opposing pattern, being more oxidized in the light, compared to the dark. In Arabidopsis trxf1f2, trxm1m2 or ntrc mutants, most protein candidates showed increased oxidation states, compared to the wild type, suggesting their light-dependent dynamics to be related to the NTRC/Trx network. In this context, deficiencies in f- and m-type Trxs were found to have different impacts on the thiol-redox proteome depending on the light environment, being higher in constant and fluctuating light, respectively, while NTRC deficiency having a strong influence in all light conditions. Results indicate the plant redox proteome to be subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. This involves f-type Trxs and NTRC to play a role in constant light conditions, while both m-type Trxs and NTRC being important to balance changes in protein redox-pattern during dynamic alterations in fluctuating light intensities.
2024-01-29 | PXD043914 | Pride
Project description:Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms. In this context, the small ubiquitous oxido-reductase thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii while much less is known about the thioredoxin interactome in this photosynthetic model organism. Using an affinity purification strategy, we identified 980 thioredoxin targets. They participate in a wide variety of metabolic pathways and cellular processes. This study broadens not only the redox regulation to new enzymes involved in metabolic pathways already known to be regulated by thioredoxins (carbon, lipid and energy metabolism) but also shed light on cellular processes for which data supporting a putative redox regulation in these processes are scarce (aromatic amino-acid biosynthesis, nuclear transport,…).
2017-07-28 | PXD006097 | Pride
Project description:The strictly anaerobic bacterium C. difficile has become one of the most problematic hospital acquired pathogens and a major burden for health care systems. Although antibiotics work effectively in most C. difficile infections (CDIs), their detrimental effect on the intestinal microbiome paves the way for recurrent episodes of CDI. To develop alternative, non-antibiotics-based treatment strategies, deeper knowledge on the physiology of C. difficile, stress adaptation mechanisms and regulation of virulence factors is mandatory. Focus of this work was to tackle the thiol proteome of C. difficile and its stress-induced alterations, since recent research reported the amino acid cysteine to play a central role in the metabolism of the pathogen. We developed a novel cysteine labeling approach to determine the redox state of protein thiols on a global scale. Applicability of the technique was demonstrated by inducing disulfide stress using the chemical diamide. The method can be transferred to any kind of redox challenge and was applied in this work to assess the effect of bile acids on the thiol proteome of C. difficile. We present redox-quantification of more than 1,500 thiol peptides and discuss the general difficulty of redox analyses of peptides possessing more than a single cysteine residue. The presented method will be especially useful when not only redox status shall be determined, but information on protein quantity is needed as well. Not least, our comprehensive dataset reveals protein cysteine sites particularly susceptible to oxidation and builds a groundwork for redox proteomics studies in C. difficile.
2018-03-05 | PXD007278 | Pride
Project description:Posttranslational modifications of protein cysteine thiols play a significant role in redox regulation and the pathogenesis of human diseases. However, the cellular redox landscape in terms of quantitative, site-specific occupancies of thiol modifications at the proteome level, especially under physiological conditions, is still largely uncharacterized. Herein, we report the site occupancies of both S-glutathionylation (SSG) and total reversible thiol oxidation (total oxidation) in RAW 264.7 macrophage cells under basal conditions. The occupancies of thiol modifications for ~4,000 cysteine sites were quantified, which revealed a mean site occupancy of 4.0% for SSG and 11.9% for total oxidation, respectively. Correlations between site occupancies and structural features such as pKa, relative residue surface accessibility, and hydrophobicity were observed. Proteome-wide site occupancy analysis also revealed a strong subcellular compartmentalization in thiol redox status, where the average occupancies of SSG and total oxidation in distinct compartments correlate well with the redox potentials of respective organelles.
2020-08-13 | PXD019913 | Pride
Project description:During infections, S. aureus has to cope with the oxidative burst of activated macrophages and neutrophils, including reactive oxygen and nitrogen species (RNS, ROS) and the strong oxidant hypochloric acid. We aimed to understand the global thiol-redox state in the major pathogen S. aureus and discover new NaOCl-sensitive proteins driven by thiol-switches. Thus, we performed a quantitative redox proteomics approach based on OxICAT and analyzed the percentages of thiol-oxidation levels in S.aureus before and after sub-lethal doses of 150 µM NaOCl stress. In parallel, we searched for protein S-bacillithiolation.
2016-12-22 | PXD004918 | Pride
Project description:Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms. In this context, the small ubiquitous oxido-reductase thioredoxin (Trx) plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii while much less is known about the thioredoxin interactome, called thioredoxome hereafter, in this photosynthetic model organism. Using a quantitative large scale proteomic analysis based on the in vitro reconstitution of the enzymatic thioredoxin system (NADPH, NTR, Trx) within an acellular extract and followed by an isotopic labeling with cleavable Isotopic-Coded Affinity Tag reagents, we identified 1052 Trx-targeted cysteines spread over 602 proteins. These Trx-targets participate in a wide variety of metabolic pathways and cellular processes. This approach, combined with the affinity purification approach study (PXD006097) broadens not only the redox regulation to new enzymes involved in metabolic pathways already known to be regulated by thioredoxins (carbon, lipid and energy metabolism) but also shed light on cellular processes for which data supporting a putative redox regulation in these processes are scarce (aromatic amino-acid biosynthesis, nuclear transport,…). Moreover, by comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteines residues.
2017-07-28 | PXD006116 | Pride
Project description:Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here, we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indiciative of disulfide stess as shown by the induction of the thiol- and oxidative stress-specific Spx, CtsR and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thiol-oxidations in response to NaOCl stress that include GapA and MetE. Shotgun-LC-MS/MS analyses revealed that GapA, Spx and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-D-phophoglycerate dehydrogenase SerA and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to up-regulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the BSH redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway.
2011-07-04 | GSE27637 | GEO