Differential Impairment of 20S and 26S Proteasome Activities in Human Hematopoietic K562 Cells during Oxidative Stress

2000 ◽  
Vol 377 (1) ◽  
pp. 65-68 ◽  
Author(s):  
Thomas Reinheckel ◽  
Oliver Ullrich ◽  
Nicolle Sitte ◽  
Tilman Grune
Keyword(s):  
Oxidative Stress ◽  
K562 Cells ◽  
26S Proteasome

scholarly journals Comparative resistance of the 20S and 26S proteasome to oxidative stress

1998 ◽  
Vol 335 (3) ◽  
pp. 637-642 ◽  
Author(s):  
Thomas REINHECKEL ◽  
Nicolle SITTE ◽  
Oliver ULLRICH ◽  
Ulrike KUCKELKORN ◽  
Kelvin J. A. DAVIES ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mammalian Cells ◽  
K562 Cells ◽  
Enzymic Activity ◽  
26S Proteasome ◽  
Proteolytic Degradation ◽  
20S Proteasome ◽  
Independent Manner ◽  
H2o2 Treatment ◽  
Fluorogenic Peptide

Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 µmol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 µmol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 µmol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 µmol of H2O2/mg, 3 µmol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2 mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1 mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20 S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.

scholarly journals Oxidative stress involvement in chemically induced differentiation of K562 cells

2000 ◽  
Vol 28 (1) ◽  
pp. 18-27 ◽  
Author(s):  
Benoı̂t Chénais ◽  
Maud Andriollo ◽  
Pascale Guiraud ◽  
Rajae Belhoussine ◽  
Pierre Jeannesson
Keyword(s):  
Oxidative Stress ◽  
K562 Cells ◽  
Induced Differentiation ◽  
Chemically Induced

scholarly journals Med13p prevents mitochondrial fission and programmed cell death in yeast through nuclear retention of cyclin C

2014 ◽  
Vol 25 (18) ◽  
pp. 2807-2816 ◽  
Author(s):  
Svetlana Khakhina ◽  
Katrina F. Cooper ◽  
Randy Strich
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Mitochondrial Fission ◽  
Regulatory System ◽  
Stress Sensitivity ◽  
26S Proteasome ◽  
Mitochondrial Fragmentation ◽  
Nuclear Retention ◽  
Cyclin C

The yeast cyclin C-Cdk8 kinase forms a complex with Med13p to repress the transcription of genes involved in the stress response and meiosis. In response to oxidative stress, cyclin C displays nuclear to cytoplasmic relocalization that triggers mitochondrial fission and promotes programmed cell death. In this report, we demonstrate that Med13p mediates cyclin C nuclear retention in unstressed cells. Deleting MED13 allows aberrant cytoplasmic cyclin C localization and extensive mitochondrial fragmentation. Loss of Med13p function resulted in mitochondrial dysfunction and hypersensitivity to oxidative stress–induced programmed cell death that were dependent on cyclin C. The regulatory system controlling cyclin C-Med13p interaction is complex. First, a previous study found that cyclin C phosphorylation by the stress-activated MAP kinase Slt2p is required for nuclear to cytoplasmic translocation. This study found that cyclin C-Med13p association is impaired when the Slt2p target residue is substituted with a phosphomimetic amino acid. The second step involves Med13p destruction mediated by the 26S proteasome and cyclin C-Cdk8p kinase activity. In conclusion, Med13p maintains mitochondrial structure, function, and normal oxidative stress sensitivity through cyclin C nuclear retention. Releasing cyclin C from the nucleus involves both its phosphorylation by Slt2p coupled with Med13p destruction.

Neuromelanin induces oxidative stress in mitochondria through release of iron: mechanism behind the inhibition of 26S proteasome

2005 ◽  
Vol 113 (5) ◽  
pp. 633-644 ◽  
Author(s):  
M. Shamoto-Nagai ◽  
W. Maruyama ◽  
H. Yi ◽  
Y. Akao ◽  
F. Tribl ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
26S Proteasome

scholarly journals Inhibition of Suicidal Erythrocyte Death by Volasertib

2017 ◽  
Vol 43 (4) ◽  
pp. 1472-1486 ◽  
Author(s):  
Abdulla Al Mamun Bhuyan ◽  
A.K.M. Ashiqul Haque ◽  
Itishri Sahu ◽  
Hang Cao ◽  
Michael S.D. Kormann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Cell Surface ◽  
Annexin V ◽  
K562 Cells ◽  
Forward Scatter ◽  
Energy Depletion ◽  
Hyperosmotic Shock ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether volasertib impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. Results: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. Conclusions: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.

scholarly journals PARAQUAT TOLERANCE3 is an E3 ligase and acts as a negative regulator of oxidative stress response

2016 ◽  
Author(s):  
Chao Luo ◽  
Xiao-Teng Cai ◽  
Jin Du ◽  
Tao-Lan Zhao ◽  
Peng-Fei Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Genetic Locus ◽  
E3 Ligase ◽  
26S Proteasome ◽  
Negative Regulator ◽  
Biotic And Abiotic Stresses ◽  
Interacting Protein ◽  
Ubiquitination Assay ◽  
Ubiquitin Ligase Activity ◽  
Arabidopsis Mutant

AbstractOxidative damage could be caused in plant cells when biotic and abiotic stresses are imposed. While the response to oxidative stress is well studied, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3) as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b may regulate the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. Moreover, PQT3 is able to recognize PRMT4b for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress.

scholarly journals Unraveling oxidative stress response in the cestode parasite Echinococcus granulosus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Martín Cancela ◽  
Jéssica A. Paes ◽  
Hercules Moura ◽  
John R. Barr ◽  
Arnaldo Zaha ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Echinococcus Granulosus ◽  
26S Proteasome ◽  
Cystic Hydatid Disease ◽  
Lamin B ◽  
Treatment And Prevention ◽  
Molecular Events ◽  
Antioxidant Proteins ◽  
Caspase Substrate ◽  
Cystic Hydatid

Abstract Cystic hydatid disease (CHD) is a worldwide neglected zoonotic disease caused by Echinococcus granulosus. The parasite is well adapted to its host by producing protective molecules that modulate host immune response. An unexplored issue associated with the parasite’s persistence in its host is how the organism can survive the oxidative stress resulting from parasite endogenous metabolism and host defenses. Here, we used hydrogen peroxide (H2O2) to induce oxidative stress in E. granulosus protoescoleces (PSCs) to identify molecular pathways and antioxidant responses during H2O2 exposure. Using proteomics, we identified 550 unique proteins; including 474 in H2O2-exposed PSCs (H-PSCs) samples and 515 in non-exposed PSCs (C-PSCs) samples. Larger amounts of antioxidant proteins, including GSTs and novel carbonyl detoxifying enzymes, such as aldo-keto reductase and carbonyl reductase, were detected after H2O2 exposure. Increased concentrations of caspase-3 and cathepsin-D proteases and components of the 26S proteasome were also detected in H-PSCs. Reduction of lamin-B and other caspase-substrate, such as filamin, in H-PSCs suggested that molecular events related to early apoptosis were also induced. We present data that describe proteins expressed in response to oxidative stress in a metazoan parasite, including novel antioxidant enzymes and targets with potential application to treatment and prevention of CHD.

4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death

2000 ◽  
Vol 113 (4) ◽  
pp. 635-641 ◽  
Author(s):  
W. Liu ◽  
M. Kato ◽  
A.A. Akhand ◽  
A. Hayakawa ◽  
H. Suzuki ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Dna Fragmentation ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
K562 Cells ◽  
Apoptotic Cell Death ◽  
Jurkat Cells ◽  
Substrate Peptide ◽  
Caspase Cascade

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20–50 μM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.

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