Imaging of Intracellular Hydrogen Peroxide Production with Hyper Upon Stimulation of Hela Cells with Egf

Markvicheva, Kseniya N.; Богданова, Е. А.; Staroverov, Dmitry B; Lukyanov, S. A.; Belousov, Vsevolod V.

doi:10.1007/978-1-59745-129-1_6

Cited by 25 publications

(8 citation statements)

References 7 publications

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“…In addition, overexpression of FLT3 ITD (but not WT FLT3) in HEK293 cells resulted in elevated fluorescence of a hydrogen peroxide (H 2 O 2 ) sensitive GFP, expressed from the vector pHyPer, 27 which was comparable with a signal induced by overexpression of the constitutively active NOX isoform NOX4 (supplemental Figure 3A). 15 Importantly, the human AML cell lines MV4-11 and MOLM13 harboring FLT3 ITD also produced high levels of ROS, whereas the AML lines THP-1, RS4-11, and EOL-1, which express WT FLT3, exhibited lower ROS levels ( Figure 2B).…”

Section: Flt3 Itd Induces High Levels Of Rosmentioning

confidence: 90%

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ

Godfrey

Arora

Bauer

et al. 2012

Blood

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Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by proteintyrosine phosphatases (PTPs). IntroductionAcute myeloid leukemia (AML) is the most frequent leukemia in adults with improving but still limited treatment possibilities, notably in elderly patients. 1,2 It arises by malignant transformation of myeloid progenitor cells. Among the contributing genetic lesions, mutations in the class III receptor tyrosine kinase (RTK) FMS-like tyrosine kinase 3 (FLT3) occur in approximately 30% of patients. 3 The prevalent type of FLT3 mutations are internal tandem duplications (ITD) of amino acid stretches in the juxtamembrane domain or in the tyrosine kinase domain, 4,5 which confer cytokineindependent proliferation and resistance to apoptosis, and causally contribute to AML in combination with additional genetic lesions. 1 Compared with the ligand-activated wild-type (WT) FLT3, FLT3 ITD mutants exhibit not only elevated but also altered signaling quality, with very pronounced activation of signal transducer and activator of transcription (STAT)5 as one characteristic feature. 6,7 FLT3 ITD also causes the production of high levels of reactive oxygen species (ROS). 8,9 Signal transduction of RTKs is regulated by protein-tyrosine phosphatases (PTPs). PTPs prevent ligand-independent RTK activation, and contribute to modulation and termination of ligand-induced signaling. 10 The activity of PTPs is regulated at several different levels. 11 One regulatory principle is the reversible oxidation of the PTP active-site cysteine, which leads to reversible inactivation. 12,13 Temporary inactivation of negatively regulating PTPs by this mechanism is believed to be important for efficient RTK signal propagation in the cell. 14 A major ROS causing cellular PTP oxidation is hydrogen peroxide (H 2 O 2 ), which can be generated by a dismutase reaction from superoxide anions, the reaction products of NADPH-oxidases. Activation of the NADPH oxidase isoform 1 (NOX1) occurs downstream of RTK activation, and involves activation and membrane translocation of the small guanosine triphosphate (GTP)ase Rac1. 15 ROS generation in the cell is counteracted by efficient ROS decomposing systems. 16 These include peroxiredoxins (Prx), which have a very low K m for H 2 O 2 and can eliminate it even at low concentrations. 17 Relatively little is known about PTPs regulating FLT3 signal transduction. We have previously shown that the nontransmembrane PTPs PTP1B and SHP-1 can potently dephosphorylate FLT3 on overexpression. Further, PTP1B appears important for suppressing signaling of newly synthesized FLT3. 7,18 SHP-2 acts as a positive regulator, because it is important for Erk activation and proliferation induced by ligand-activated WT FLT3. However, it is dispensable for FLT3 ITD-mediated transformation. 19 We previously performed a shRNA-based screen to identify PTPs regulating WT FLT3. The initial screen assessed the effects of shRNAs for 20 PTPs on FL-induced Erk1/2 activation in WT FLT3-expressing 32D cells. Among several potentia...

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Section: Flt3 Itd Induces High Levels Of Rosmentioning

confidence: 90%

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ

Godfrey

Arora

Bauer

et al. 2012

Blood

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“…Forty microliters of cells was placed on a Polysine microscope slide (25 by 75 by 1 mm; Thermo Scientific, Waltham, MA), covered with a Fisher-brand microscope glass coverslip (diameter, 15 mm; thickness, 0.13 to 0.17 mm; Thermo Scientific), and then visualized under a confocal laser scanning microscope (Leica model TCS SP8; Leica Microsystems, Buffalo Grove, IL, USA). Excitation was provided at 488 nm, with emission being collected from a wavelength range of 500 to 530 nm (32,56). For each sample, at least 5 fluorescent and differential interference contrast (DIC) images were captured.…”

Section: Methodsmentioning

confidence: 99%

Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

et al. 2020

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Preexposure to a low concentration of H2O2 significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H2O2. However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 μM H2O2 and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn2+-coordinated Cys139 and Cys142 mutations eliminated the H2O2 oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn2+ in H2O2-treated PerR, demonstrating that cysteine oxidation results in Zn2+ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn2+-bound PerR protein (PerR:Zn,Mn) was abolished by H2O2 treatment but was restored by dithiothreitol reduction, verifying that H2O2 inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H2O2-concentration-induced adaptation of S. oligofermentans to a higher H2O2 concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H2O2 concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H2O2 defense strategy employed by catalase-negative streptococci in Mn2+-rich cellular environments. IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.

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“…Given this, one can speculate that either the intracellular level of RNOS is in the submicromolar range or an intracellular oxidative burst with a rapid decay occurs that cannot be adequately measured using current technology. Levels of RNOS generation may be clarified by novel reversible redox-sensitive fluorescent proteins (19,45,106,111) or mass spectrometry approaches that may allow for the accurate and sensitive quantification of oxidants at the organelle level (38). Despite the low levels of RNOS suggested by these measurements, modulating the expression levels of NOXs, NOS, superoxide dismutases (SODs), or catalase, in vitro and in vivo (56,61), proves that endogenous RNOS play an important role in multiple cellular functions.…”

Section: Cellular Sources and Levels Of Rnosmentioning

confidence: 99%

Regulation of Cell Physiology and Pathology by Protein S-Glutathionylation: Lessons Learned from the Cardiovascular System

Pimentel

Haeussler²,

Matsui³

et al. 2012

Antioxidants & Redox Signaling

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Significance: Reactive oxygen and nitrogen species contributing to homeostatic regulation and the pathogenesis of various cardiovascular diseases, including atherosclerosis, hypertension, endothelial dysfunction, and cardiac hypertrophy, is well established. The ability of oxidant species to mediate such effects is in part dependent on their ability to induce specific modifications on particular amino acids, which alter protein function leading to changes in cell signaling and function. The thiol containing amino acids, methionine and cysteine, are the only oxidized amino acids that undergo reduction by cellular enzymes and are, therefore, prime candidates in regulating physiological signaling. Various reports illustrate the significance of reversible oxidative modifications on cysteine thiols and their importance in modulating cardiovascular function and physiology. Recent Advances: The use of mass spectrometry, novel labeling techniques, and live cell imaging illustrate the emerging importance of reversible thiol modifications in cellular redox signaling and have advanced our analytical abilities. Critical Issues: Distinguishing redox signaling from oxidative stress remains unclear. S-nitrosylation as a precursor of S-glutathionylation is controversial and needs further clarification. Subcellular distribution of glutathione (GSH) may play an important role in local regulation, and targeted tools need to be developed. Furthermore, cellular redundancies of thiol metabolism complicate analysis and interpretation. Future Directions: The development of novel pharmacological analogs that specifically target subcellular compartments of GSH to promote or prevent local protein S-glutathionylation as well as the establishment of conditional gene ablation and transgenic animal models are needed. Antioxid. Redox Signal. 16, 524-542.

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Imaging of Intracellular Hydrogen Peroxide Production with Hyper Upon Stimulation of Hela Cells with Egf

Cited by 25 publications

References 7 publications

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ

Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

Regulation of Cell Physiology and Pathology by Protein S-Glutathionylation: Lessons Learned from the Cardiovascular System

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