Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes.
ABSTRACT: Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment.
Project description:Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA-mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1? (HIF-1?) or the binding of HIF-1? to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1? binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.
Project description:Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts.
Project description:Melatonin (N-acetyl-5-methoxytryptamine) is synthesized from tryptophan by Saccharomyces cerevisiae and non-conventional yeast species. Antioxidant properties have been suggested as a possible role of melatonin in a S. cerevisiae wine strain. However, the possible antioxidant melatonin effect on non-Saccharomyces species and other strains of S. cerevisiae must be evaluated. The aim of this study was to determine the antioxidant capacity of melatonin in eight S. cerevisiae strains and four non-conventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Starmerella bacillaris, and Hanseniaspora uvarum). Therefore, the ROS formation, lipid peroxidation, catalase activity, fatty acid composition, and peroxisome proliferation were investigated. The results showed that the presence of melatonin increases peroxisome accumulation and slightly increases the catalase activity. When cells grown in the presence of melatonin were exposed to oxidative stress induced by H2O2, lower ROS accumulation and lipid peroxidation were observed in all tested strains. Therefore, the increased catalase activity that was a consequence of oxidative stress was lower in the presence of melatonin. Moreover, the presence of MEL modulates cell FA composition, increasing oleic and palmitoleic acids and leading to higher UFA/SFA ratios, which have been previously related to a higher tolerance to H2O2. These findings demonstrate that melatonin can act as an antioxidant compound in both S. cerevisiae and non-Saccharomyces yeasts.
Project description:Non-enzymatic lipid peroxidation of the skin-lipid bilayer causes perturbations that affect the biomembrane structure, function, and permeability of reactive oxygen species (ROS). In the present study, we employed molecular dynamics simulations to study the effect of lipid peroxidation on the bilayer structural properties and permeability of various ROS. The oxidized skin-lipid bilayer was composed of ceramide, cholesterol, free fatty acid, and 5?-hydroperoxycholesterol (5?-CH). The simulation showed that, upon oxidation, the oxidized group (-OOH) of 5?-CH migrates towards the aqueous phase and the backbone of 5?-CH tilts, which causes the membrane to expand laterally. Measurements of the permeability of all ROS along the oxidized skin-lipid bilayer revealed a decreased breaching barrier for all the species as the degree of peroxidation increased, with a resulting easy passage across the membrane. The insights from the simulations indicate that lipid peroxidation might perturb the membrane barrier, thereby inflicting oxidative stress that leads to apoptosis. This study helps to understand oxidative stress at the atomic level. To our knowledge, this is the first reported molecular dynamics simulation study on oxidized skin-lipid bilayer and permeability of ROS.
Project description:Lipid peroxidation by reactive oxygen species (ROS) during oxidative stress is non-enzymatic damage that affects the integrity of biological membrane, and alters the fluidity and permeability. We conducted molecular dynamic simulation studies to evaluate the structural properties of the bilayer after lipid peroxidation and to measure the permeability of distinct ROS. The oxidized membrane contains free fatty acid, ceramide, cholesterol, and 5?-hydroperoxycholesterol (5?-CH). The result of unconstrained molecular dynamic simulations revealed that lipid peroxidation causes area-per-lipid of the bilayer to increase and bilayer thickness to decrease. The simulations also revealed that the oxidized group of 5?-CH (-OOH) moves towards the aqueous layer and its backbone tilts causing lateral expansion of the bilayer membrane. These changes are detrimental to structural and functional properties of the membrane. The measured free energy profile for different ROS (H<sub>2</sub>O<sub>2</sub>, HO<sub>2</sub>, HO, and O<sub>2</sub>) across the peroxidized lipid bilayer showed that the increase in lipid peroxidation resulted in breaching barrier decrease for all species, allowing easy traversal of the membrane. Thus, lipid peroxidation perturbs the membrane barrier and imposes oxidative stress resulting into apoptosis. The collective insights increase the understanding of oxidation stress at the atomic level.
Project description:Drought is an abiotic stress that affects plant growth, and lipids are the main economic factor in the agricultural production of oil crops. However, the molecular mechanisms of drought response function in lipid metabolism remain little known. In this study, overexpression (OE) of different copies of the drought response genes LEA3 and VOC enhanced both drought tolerance and oil content in Brassica napus and Arabidopsis. Meanwhile, seed size, membrane stability and seed weight were also improved in OE lines. In contrast, oil content and drought tolerance were decreased in the AtLEA3 mutant (atlea3) and AtVOC-RNAi of Arabidopsis and in both BnLEA-RNAi and BnVOC-RNAi B. napus RNAi lines. Hybrids between two lines with increased or reduced expression (LEA3-OE with VOC-OE, atlea3 with AtVOC-RNAi) showed corresponding stronger trends in drought tolerance and lipid metabolism. Comparative transcriptomic analysis revealed the mechanisms of drought response gene function in lipid accumulation and drought tolerance. Gene networks involved in fatty acid (FA) synthesis and FA degradation were up- and down-regulated in OE lines, respectively. Key genes in the photosynthetic system and reactive oxygen species (ROS) metabolism were up-regulated in OE lines and down-regulated in atlea3 and AtVOC-RNAi lines, including LACS9, LIPASE1, PSAN, LOX2 and SOD1. Further analysis of photosynthetic and ROS enzymatic activities confirmed that the drought response genes LEA3 and VOC altered lipid accumulation mainly via enhancing photosynthetic efficiency and reducing ROS. The present study provides a novel way to improve lipid accumulation in plants, especially in oil production crops.
Project description:To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis.Rat hepatoma FaO cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations (25 to 100 ?mol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays.Lipid-loading resulted in intracellular triglyceride (TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein-1c (SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species (ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B (NF-?B). Incubation of steatotic cells with silybin 50 ?mol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1 (CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-?B activation.We demonstrated the direct anti-steatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies.
Project description:Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. Here, we find that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids are peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delays the onset of neurodegeneration. Furthermore, a similar pathway leads to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease.
Project description:Ferroptosis is a novel form of non-apoptotic regulated cell death (RCD), with distinct characteristics and functions in physical conditions and multiple diseases such as cancers. Unlike apoptosis and autophagy, this new RCD is an iron-dependent cell death with features of lethal accumulation of reactive oxygen species (ROS) and over production of lipid peroxidation. Excessive iron from aberrant iron metabolisms or the maladjustment of the two main redox systems thiols and lipid peroxidation role as the major causes of ROS generation, and the redox-acrive ferrous (intracellular labile iron) is a crucial factor for the lipid peroxidation. Regulation of ferrroptosis also involves different pathways such as mevalonate pathway, P53 pathway and p62-Keap1-Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. Ferroptosis roles as a double-edged sword either suppressing or promoting tumor progression with the release of multiple signaling molecules in the tumor microenvironment. Emerging evidence suggests ferroptosis as a potential target for cancer therapy and ferroptosis inducers including small molecules and nanomaterials have been developed. The application of ferroptosis inducers also relates to overcoming drug resistance and preventing tumor metastasis, and may become a promising strategy combined with other anti-cancer therapies. Here, we summarize the ferroptosis characters from its underlying basis and role in cancer, followed by its possible applications in cancer therapies and challenges maintained.
Project description:Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus of the Flaviviridae family. HCV-infected hepatocytes are known to produce reactive oxygen species (ROS), which initiate lipid peroxidation, a reaction that converts polyunsaturated fatty acids, such as arachidonate, into reactive carbonyls that inactivate proteins. To study the effect of lipid peroxidation on HCV replication, we administered arachidonate to Huh7 cells that harbor an HCV replicon (Huh7-K2040 cells). After incubation in medium supplemented with arachidonate but deprived of lipid-soluble antioxidants, the cellular amount of malondialdehyde (MDA), a product of lipid peroxidation, increased markedly in Huh7-K2040 cells but not in parental Huh7 cells that do not harbor an HCV replicon. This increase was followed by a sharp reduction (>95%) in HCV RNA. Both of these events were prevented when cells were treated with vitamin E, a lipid-soluble antioxidant. After prolonged incubation of Huh7-K2040 cells with arachidonate in the absence of lipid-soluble antioxidants, the amount of MDA decreased after the reduction in the amount of HCV RNA. Thus, in the presence of arachidonate and in the absence of lipid-soluble antioxidants, HCV replication induces lipid peroxidation that reduces the amount of HCV RNA. Our results provide a mechanism for the previous observation that polyunsaturated fatty acids inhibit HCV replication [Kapadia SB, Chisari FV (2005) Proc Natl Acad Sci USA 102:2561-2566], and they suggest that these agents may be effective in inhibiting HCV replication in vivo.