ABSTRACT
Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving the chromatin DNA. The molecular mechanism of EndoG still remains unknown in higher organisms. Here, we determined the crystal structure of mouse EndoG at â¼1.96 Å resolution. The EndoG shows an altered dimeric configuration in which N-terminal region of one subunit interact to the other subunit in dimer. The deletion of this region that is highly conserved in mammalian EndoGs resulted in a monomer with significantly reduced activity suggesting the association of the dimeric arrangement into the nuclease activity. Furthermore, we observed a large conformational change in the loop of the active site groove in EndoG, which corresponds to the DNA binding region. Intriguingly, EndoG dimers are linked by oxidation of the reactive cysteine 110 in this flexible loop to form a long oligomeric chain in the crystal lattice. The structural analysis and ensuing biochemical data suggest that this flexible loop region in the active site is important to the regulation of EndoG nuclease function in mouse.
Subject(s)
Endodeoxyribonucleases/chemistry , Animals , Catalytic Domain , Crystallography, X-Ray , Cysteine/metabolism , DNA/chemistry , Mice , Models, Molecular , Oxidation-Reduction , Protein Binding , Protein MultimerizationABSTRACT
Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP+ -dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.
Subject(s)
Apoptosis , Isocitrate Dehydrogenase/chemistry , Lipid Peroxidation , NADP/chemistry , Oxalates/pharmacology , Amidines/chemistry , Antioxidants/pharmacology , Binding, Competitive , Cell Separation , Cytosol/metabolism , DNA/chemistry , Flow Cytometry , Glutathione/metabolism , Humans , Immunoblotting , Microscopy, Fluorescence , Oxalates/chemistry , Oxidants/pharmacology , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species , Time Factors , U937 CellsABSTRACT
Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a prooxidant condition.
Subject(s)
Enzyme Inhibitors/pharmacology , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/metabolism , Lipid Peroxidation/physiology , NADP/metabolism , Aldehydes/metabolism , Aldehydes/pharmacology , Enzyme Inhibitors/metabolism , Glutathione/metabolism , Humans , Lipid Peroxidation/drug effects , Lipid Peroxides/metabolism , Lipid Peroxides/pharmacology , Malondialdehyde/metabolism , Malondialdehyde/pharmacology , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Time Factors , U937 CellsABSTRACT
Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) in U937 cells produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against lipid peroxidation-mediated oxidative damage in U937 cells was investigated in control cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the viability was lower and the protein oxidation, lipid peroxidation, and oxidative DNA damage, reflected by an increase in 8-hydroxy-2'-deoxyguanosine, were higher in oxalomalate-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2',7'-dichlorodihydrofluorescin, as well as the significant decrease in the intracellular GSH level in oxalomalate-treated U937 cells upon exposure to AAPH. These results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against lipid peroxidation-mediated oxidative damage through the removal of reactive oxygen species.
Subject(s)
Enzyme Inhibitors/pharmacology , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/metabolism , Lipid Peroxidation/drug effects , NADP/metabolism , Oxalates/pharmacology , Amidines/pharmacology , Cells, Cultured , Glutathione/metabolism , Humans , Lipid Peroxidation/physiology , Malondialdehyde/analysis , Malondialdehyde/metabolism , Microscopy, Confocal/methods , Organophosphorus Compounds/analysis , Organophosphorus Compounds/metabolism , Oxidants/pharmacology , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Pyrenes/analysis , Pyrenes/metabolism , U937 CellsABSTRACT
Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. The oxyR gene product regulates the expression of enzymes and proteins that are needed for cellular protection against oxidative stress. Upon exposure to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the Escherichia coli oxyR overexpression mutant was much more resistant to lipid peroxidation-mediated cellular damage, when compared to the OxyR deletion mutant in regard to growth kinetics, viability, and DNA damage. The deletion of the OxyR gene in E. coli also resulted in increased susceptibility of superoxide dismutase to lipid peroxidation-mediated inactivation. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in free radical-induced cellular damage. Also, the oxyR regulon plays an important protective role in lipid peroxidation-mediated cellular damage.
Subject(s)
Bacterial Proteins/pharmacology , DNA-Binding Proteins , Deoxyguanosine/analogs & derivatives , Escherichia coli/genetics , Lipid Peroxidation/drug effects , Oxidative Stress/drug effects , Regulon , Repressor Proteins/pharmacology , Transcription Factors/pharmacology , 8-Hydroxy-2'-Deoxyguanosine , Amidines/pharmacology , Bacterial Proteins/genetics , DNA/metabolism , Deoxyguanosine/metabolism , Escherichia coli Proteins , Free Radicals , Gene Expression Regulation, Bacterial , Mutagens/pharmacology , Oxidants/pharmacology , Oxidation-Reduction , Repressor Proteins/genetics , Superoxide Dismutase/metabolism , Transcription Factors/geneticsABSTRACT
Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. In the present study, we evaluated lipid peroxidation-mediated cytotoxicity and oxidative DNA damage in U937 cells. Upon exposure of U937 cells to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the cells exhibited a reduction in viability and an increase in the endogenous production of reactive oxygen species (ROS), as measured by the oxidation of 2',7'-dichlorodihydrofluorescein. In addition, a significant decrease in the intracellular GSH level and the activities of major antioxidant enzymes were observed. We also observed lipid peroxidation-mediated oxidative DNA damage, reflected by an increase in 8-OH-dG level and loss of the ability of DNA to renature. When the cells were pretreated with the antioxidant N-acetylcysteine (NAC) or the spin trap alpha-phenyl-N-t-butylnitrone (PBN), lipid peroxidation-mediated cytotoxicity in U937 cells was protected. This effect seems to be due to the ability of NAC and PBN to reduce ROS generation induced by lipid peroxidation. These results suggest that lipid peroxidation resulted in a pro-oxidant condition of U937 cells by the depletion of GSH and inactivation of antioxidant enzymes, which consequently leads to a decrease in survival and oxidative damage to DNA. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in oxidative stress-induced cellular damage.
