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Redox proteomic analysis of H2O2 -treated Jurkat cells and effects of bicarbonate and peroxiredoxins 1 and 2

ABSTRACT: Oxidation of thiol proteins and redox signaling occur in cells exposed to H2O2 but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of H2O2 with CO2/bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdx1 or Prdx2). We measured changes in oxidation state of specific Cys-SH residues on treating Jurkat T cells with H2O2,by isotopically labeling reduced thiols and analyzing the tagged peptides by mass spectrometry. Cells treated in the absence and presence of bicarbonate were compared, as well as wildtype cells and cells in which Prdx1 or Prdx2 had been knocked out.

ORGANISM(S): Homo Sapiens

SUBMITTER: Jing Yang

PROVIDER: PXD053738 | iProX | Mon Jul 08 00:00:00 BST 2024

REPOSITORIES: iProX

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Redox proteomic analysis of H2O2 -treated Jurkat cells and effects of bicarbonate and knockout of peroxiredoxins 1 and 2.

Pace Paul E PE Fu Ling L Hampton Mark B MB Winterbourn Christine C CC

Free radical biology & medicine 20241101

Oxidation of thiol proteins and redox signaling occur in cells exposed to H2O2 but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of H2O2 with CO2/bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdxs). Changes in oxidation state of specific Cys-SH residues on treating Jurkat T lymphoma cells with ...[more]

PMID: 39489196

Similar Datasets

Thiol redox proteomic changes in Jurkat cells resulting from knockout of peroxiredoxin 1 or peroxiredoxin 2

Project description:Peroxiredoxins are thiol proteins involved in antioxidant defense and redox signaling. One putative signaling mechanism involves the facilitated oxidation of associated proteins via a redox relay. We have investigated if removal of the cytoplasmic peroxiredoxins Prdx1 and Prdx2 influences the redox state of other thiol proteins in cultured cells. A mass spectrometric proteomic approach in which reduced thiols in one cell type were labelled with a heavy isotope tag and reduced thiols in the other cell type with a light isotope tag, was used to compare wild type (WT) Jurkat cells with cells in which either Prdx1 or Prdx2 was knocked out (KO).

2024-07-02 | PXD053545 |

The Human 2-Cys Peroxiredoxins Form Widespread, Cysteine-Dependent- And Isoform-Specific Protein-Protein Interactions.

Project description:Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 reacts poorly with most cysteine thiols and it is not clear how it discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins. It was suggested that peroxiredoxins act as peroxidases to facilitate H2O2-dependent oxidation of proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of peroxiredoxins, we show that all five human 2-cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this precedence. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses.

2021-05-04 | PXD024114 | Pride

Peroxiredoxin 2 is required for the exercise induced redox mediated adaptation to exercise

Project description:During exercise there is a site-specific increase in ROS within muscle required for the beneficial adaptive response by activation of specific signalling pathways. Peroxiredoxin 2 (Prdx2), an abundant cytoplasmic 2-Cys peroxidase, is required for the adaptive beneficial hormesis response to H2O2, myoblasts with knockdown of Prdx2 did not increase mitochondrial content and had disrupted myogenesis. Using a 5-day swimming protocol in C. elegans, previously been demonstrated to improve healthspan, we observed increased mitochondrial content, fitness, survival and longevity in wild type (N2) worms. However, prdx-2 mutant worms had decreased fitness, disrupted mitochondria, reduced survival and lifespan following exercise. Global proteomics following exercise revealed an increase in the abundance of proteins involved in fatty oxidation and ion transport, decrease in proteins involved in cuticle formation in N2 worms. In the prdx-2 mutants following exercise there was an increase in proteins related to the mTOR pathway and decrease in transsulfuration and microRNA biogenesis proteins. Relative quantification of the reversible oxidation state of individual Cysteine (Cys) residues revealed an increased oxidation of specific Cys residues following exercise in prdx-2 mutant. A number of regulatory Cys residues of metabolic, cytoskeletal and calcium handling proteins are reduced in N2 worms. A redox signalling relay whereby the oxidative equivalents from ROS are transferred from evolutionary conserved Prdxs to target proteins, would confer specificity on redox signalling required for the beneficial adaptations to exercise.

2023-03-11 | PXD037698 | Pride

Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

Project description:In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs). While regulation by activation segment phosphorylation in Ser/Thr kinases is well understood, relatively little is known about how oxidation of cysteine (Cys) amino acids modulates catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif, a highly conserved T-Loop Cys and a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

2025-04-01 | PXD044990 | Pride

Peroxisome-derived hydrogen peroxide can modulate the sulfenylation profiles of key redox signaling proteins in Flp-In T-REx 293 cells

Project description:Ever since the first characterization of peroxisomes, a central theme has been their involvement in cellular hydrogen peroxide (H2O2) metabolism. While the reputation of H2O2 drastically changed from an exclusively toxic molecule to a signaling messenger, the regulatory role of peroxisomes in these signaling events is still largely underappreciated. This is mainly because the number of known protein targets of peroxisome-derived H2O2 is rather limited and testing of specific targets is predominantly based on knowledge previously gathered in related fields of research. To gain a broader and more systematic insight into the role of peroxisomes in redox signaling, new approaches are urgently needed. In this study, we have combined a previously developed Flp-In T-REx 293 cell system in which peroxisomal H2O2 production can be modulated with a yeast AP-1-like-based sulfenome mining strategy to inventory protein thiol targets of peroxisome-derived H2O2 in different subcellular compartments. Using this approach, we were able to identify specific and common targets of peroxisome-derived and exogenous H2O2 in peroxisomes, the cytosol, and mitochondria. We also observed that the sulfenylation kinetics profiles of key targets (e.g., PRDX1, ANXA2, and TUBA1C) belonging to different protein families (e.g., antioxidants, annexins, and tubulins) can vary considerably. In addition, we obtained compelling but indirect evidence that peroxisome-derived H2O2 may oxidize at least some of its targets (e.g., transcription factors) through a redox relay mechanism. In conclusion, given that sulfenic acids function as key intermediates in H2O2 signaling, the findings presented in this study provide initial but critical insight into how peroxisomes may be integrated into the cellular H2O2 signaling network.

2022-05-10 | PXD030782 | Pride

Genome-Wide Study Based On Abasic Site Revealed Selective RNA Oxidation In Neuro 2a Cells

Project description:To investigate selectivity of mRNA oxidation, total RNA and oxidized RNA isolated from Neuro 2a cells before and after H2O2 treatment were employed for microarray analysis. It was found that selective oxidation of mRNA already occurs under normal culture conditions but was increased by H2O2, especially in a subset of mRNAs related to certain functions. Moreover, mRNA oxidation level is also related to its abundance or stability (half-life time). This shows for the first time that mRNA oxidation is associated with RNA homeostasis including function, stability and abundance depending on cellular redox status in a genome-wide scale.

2012-10-01 | GSE39641 | GEO

MOF-mediated PRDX1 acetylation regulates LPS-induced macrophage activation.

Project description:Macrophages respond to environmental cues in a plastic manner and play a crucial role in host defense, inﬂammation, and tissue homeostasis. Macrophage activation undergoes metabolic and transcriptomic reprogramming to adapt and tailor an appropriate response, regulating inﬂammation. Reactive oxygen species (ROS) have been shown to play a pivotal role in macrophage activation. Nevertheless, the detailed molecular mechanism of how initial redox signals direct macrophage activation is not fully elucidated. Here, we uncover an unconventional role for histone acetyltransferase MOF (also known as KAT8) in regulating LPS-induced macrophage activation via modulating the acetylation of peroxiredoxin 1 (PRDX1), an H2O2 scavenger. We demonstrate that PRDX1 is a novel substrate of MOF and identify the major MOF-mediated acetylation site at lysine 197. K197 acetylation of PRDX1 (K197ac PRDX1) is dynamic and reversely regulated by HDAC6 or SIRT2. We observe that this acetylation is readily diminished in the immediate response to LPS. We demonstrate that K197ac PRDX1 fine-tunes its peroxidase activity, leading to the modulation of intracellular ROS levels in early response to LPS stimulation. Moreover, K197ac PRDX1 specifically regulates ERK1/2 phosphorylation, thereby modulating glycolytic metabolism in inflammatory macrophages. Consequently, K197ac PRDX1 tunes the expression and production of the proinflammatory cytokine, IL-6. Taken together, our findings describe a novel signaling module, MOF-PRDX1-ERK, which regulates LPS-induced macrophage activation at the metabolic and transcriptional levels.

2024-12-31 | GSE231941 | GEO

Glycine max

Project description:H2O2-induced oxidation at GmNTL1 Cys-247 is involved in plant salt tolerance

| PRJNA1057058 | ENA

Identification of the S-sulfenylated residues of GSNOR1 in Arabidopsis

Project description:The major mechanism by which H2O2 executes its physiological functions is through oxidation of sulfur in the target proteins. Thiol groups of protein cysteine (Cys) could be oxidized by H2O2 to sulfenic, a process also known as S-sulfenylation. Therefore, we reasoned that the activity of GSNOR1 might be regulated by H2O2 through S-sulfenylation. An in vitro S-sulfenylation assay revealed that GSNOR1-His recombinant protein was S-sulfenylated. Because Cys-284 and Cys-285 of GSNOR1 are adjacent and located on the same enzymolysis peptide segment, and also because both of them were identified as S-sulfenylated residues in vivo, to further test whether Cys-284 is S-sulfenylated, Cys-285 was substituted with serine (Ser).

2025-01-23 | PXD056375 | Pride

Concomitant inhibition of the thioredoxin system and non-homologous DNA repair potently sensitizes Philadelphia-positive lymphoid leukemia to tyrosine kinase inhibitors

Project description:BCR-ABL1 gene fusion is an essential driver lesion in both chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). While tyrosine kinase inhibitors (TKIs) cure up to 95% of CML patients, 50 % of Ph+ B-ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B-ALL. Dysregulated redox homeostasis manifested by the increased reactive oxygen species (ROS) levels and the upregulation of the thioredoxin (TXN) antioxidant system has been previously shown in B-ALL. Pharmacological inhibition of the TXN system with auranofin (AUR) efficiently kills B-ALL cells, indicating that the TXN system plays a major role in maintaining redox homeostasis in B-ALL cells. Here we show that peroxiredoxin-1 (PRDX1), one of the enzymes of the TXN system responsible for scavenging H2O2, is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. The genomic knockout of PRDX1 negatively affects the viability of Ph+ B-ALL cells and sensitizes them to TKIs, while no such effects occur in myeloid cells. By analyzing global changes in gene expression in PRDX1-deficient cells treated with imatinib we discover non-homologous end-joining (NHEJ) DNA repair pathways as a novel vulnerability of Ph+ B-ALL cells. Accordingly, pharmacologic inhibition of the TXN system diminishes the viability of Ph+ B-ALL cells and enhances their sensitivity to TKIs and NHEJ inhibitors. Finally, we propose a novel combination of TKIs, TXN inhibitors, and NHEJ inhibitors as a potential therapeutic approach for the treatment of Ph+ B-ALL.

2024-03-01 | GSE221906 | GEO