Plasma membrane redox system protects cells against oxidative stress

Abstract

Aerobic organisms have developed defensive systems to survive in the presence of oxygen and its highly reactive species (ROS). The cellular mechanisms of protection against oxidative injury include: (i) specific enzymes, such as catalase, glutathione peroxidase and superoxide dismutase; (ii) small hydrophilic molecules, such as ascorbate, glutathione and uric acid; and (iii) hydrophobic agents, such as ubiquinone and α-tocopherol in membranes.¹ Among these, coenzyme Q (CoQ) is the only lipid-soluble antioxidant that can be synthesized in all organisms so far studied.     

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H(2)O(2), tert-butyl hydroperoxide, and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Delta(1)-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels, and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H(2)O(2) levels, increased endogenous P5CS and P5CR expression was observed, indicating that upregulation of proline biosynthesis is an oxidative stress response.     

Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress

Mitochondria combine the production of energy with an efficient chain of reduction–oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.     

Quercetin protects HCT116 cells from Dichlorvos-induced oxidative stress and apoptosis

The present study was designed to assess the possible protective effects of Quercetin (QUER), a flavonoid with well-known pharmacological effects, against Dichlorvos (DDVP)-induced toxicity in vitro using HCT116 cells. The cytotoxicity was monitored by cell viability, reactive oxygen species (ROS) generation, anti-oxidant enzyme activities, malondialdehyde (MDA) production, and DNA fragmentation. The apoptosis was assessed through the measurement of the mitochondrial transmembrane potential (ΔΨm) and caspase activation. The results indicated that pretreatment of HCT116 cells with QUER, 2 h prior to DDVP exposure, significantly decreased the DDVP-induced cell death, inhibited the ROS generation, modulated the activities of catalase (CAT) and superoxide dismutase (SOD), and reduced the MDA level. The reductions in mitochondrial membrane potential, DNA fragmentation, and caspase activation were also attenuated by QUER. These findings suggest that dietary QUER can protect HCT116 cells against DDVP-induced oxidative stress and apoptosis.     

Autotaxin protects microglial cells against oxidative stress

Oxidative stress occurs when antioxidant defenses are overwhelmed by oxygen-reactive species and can lead to cellular damage, as seen in several neurodegenerative disorders. Microglia are specialized cells in the central nervous system that act as the first and main form of active immune defense in the response to pathological events. Autotaxin (ATX) plays an important role in the modulation of critical cellular functions, through its enzymatic production of lysophosphatidic acid (LPA). In this study, we investigated the potential role of ATX in the response of microglial cells to oxidative stress. We show that treatment of a microglial BV2 cell line with hydrogen peroxide (H(2)O(2)) stimulates ATX expression and LPA production. Stable overexpression of ATX inhibits microglial activation (CD11b expression) and protects against H(2)O(2)-treatment-induced cellular damage. This protective effect of ATX was partially reduced in the presence of the LPA-receptor antagonist Ki16425. ATX overexpression was also associated with a reduction in intracellular ROS formation, carbonylated protein accumulation, proteasomal activity, and catalase expression. Our results suggest that up-regulation of ATX expression in microglia could be a mechanism for protection against oxidative stress, thereby reducing inflammation in the nervous system.     

Solution structure of the SWIRM domain of human histone demethylase LSD1

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.     

Quiescence induced by iron challenge protects neuroblastoma cells from oxidative stress

The brain uses massive amounts of oxygen, generating large quantities of reactive oxygen species (ROS). Because of its lipid composition, rich in unsaturated fatty acids, the brain is especially vulnerable to ROS. Furthermore, oxidative damage in the brain is often associated with iron, which has pro-oxidative properties. Iron-mediated oxidative damage in the brain is compounded by the fact that brain iron distribution is non-uniform, being particularly high in areas sensitive to neurodegeneration. This work was aimed to further our understanding of the cellular mechanisms by which SHSY5Y neuroblastoma cells adapt to, and survive increasing iron loads. Using an iron accumulation protocol that kills about 50% of the cell population, we found by cell sorting analysis that the SHSY5Y sub-population that survived the iron loading arrested in the G(0) phase of the cell cycle. These cells expressed neuronal markers, while their electrical properties remained largely unaltered. These results suggest that upon iron challenge, neuroblastoma cells respond by entering the G(0) phase, somehow rendering them resistant to oxidative stress. A similar physiological condition might be involved in neuronal survival in tissues known to accumulate iron with age, such as the hippocampus and the substantia nigra pars compacta.     

Lipoamide protects retinal pigment epithelial cells from oxidative stress and mitochondrial dysfunction

alpha-Lipoic acid (LA) has been widely studied as an agent for preventing and treating various diseases associated with oxidative disruption of mitochondrial functions. To investigate a related mitochondrial antioxidant, we compared the effects of lipoamide (LM), the neutral amide of LA, with LA for measures of oxidative damage and mitochondrial dysfunction in a human retinal pigment epithelial (RPE) cell line. Acrolein, a major component of cigarette smoke and a product of lipid peroxidation, was used to induce oxidative mitochondrial damage in RPE cells. Overall, using comparable concentrations, LM was more effective than LA at preventing acrolein-induced mitochondrial dysfunction and oxidative stress. Relative to LA, LM improved ATP levels, membrane potentials, and activities of mitochondrial complexes I, II, and V and dehydrogenases that had been decreased by acrolein exposure. LM reduced acrolein-induced oxidant generation, calcium levels, protein oxidation, and DNA damage to a greater degree than LA. And, total antioxidant capacity, glutathione content, glutathione S-transferase, and superoxide dismutase activities and expression of nuclear factor-E2-related factor 2 were increased by LM relative to LA. These results suggest that LM is a more potent mitochondrial-protective agent and antioxidant than LA in protecting RPE from oxidative damage.     

Superoxide dismutase 1 protects retinal cells from oxidative damage

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.     

ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis

The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b^−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b^−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.     

Pink1 protects cortical neurons from thapsigargin-induced oxidative stress and neuronal apoptosis

Apoptosis mediates the precise and programmed natural death of neurons and is a physiologically important process in neurogenesis during maturation of the central nervous system. However, premature apoptosis and/or an aberration in apoptosis regulation are implicated in the pathogenesis of neurodegeneration. Thus, it is important to identify neuronal pathways/factors controlling apoptosis. Pink1 [phosphatase and tensin homologue (PTEN)-induced kinase 1] is a ubiquitously expressed gene and has been reported to have a physiological role in mitochondrial maintenance, suppressing mitochondrial oxidative stress, fission and autophagy. However, how Pink1 is involved in neuronal survival against oxidative stress remains not well understood. In the present paper, we demonstrate that thapsigargin, a specific irreversible inhibitor of endoplasmic reticulum (ER) calcium-ATPase, could lead to dramatic oxidative stress and neuronal apoptosis by ectopic calcium entry. Importantly, the neuronal toxicity of thapsigargin inhibits antioxidant gene Pink1 expression. Although Pink1 knockdown enhances the neuronal apoptosis by thapsigargin, its overexpression restores it. Our findings have established the neuronal protective role of Pink1 against oxidative stress and afford rationale for developing new strategy to the therapy of neurodegenerative diseases.     

Hydroxytyrosol protects retinal pigment epithelial cells from acrolein-induced oxidative stress and mitochondrial dysfunction

Hydroxytyrosol (HTS) is a natural polyphenol abundant in olive oil. Increasing evidence indicates HTS has beneficial effect on human health for preventing various diseases. In the present study, we investigated the protective effects of HTS on acrolein-induced toxicity in human retinal pigment epithelial cell line, ARPE-19, a cellular model of smoking- and age-related macular degeneration. Acrolein, a major component of the gas phase cigarette smoke and also a product of lipid peroxidation in vivo , at 75 μmol/L for 24 h caused significant loss of cell viability, oxidative damage (increase in oxidant generation and oxidative damage to proteins and DNA, decrease in antioxidants and antioxidant enzymes, and also inactivation of the Keap1/Nrf2 pathway), and mitochondrial dysfunction (decrease in membrane potential, activities of mitochondrial complexes, viable mitochondria, oxygen consumption, and factors for mitochondrial biogenesis, and increase in calcium). Pre-treatment with HTS dose dependently and also time dependently protected the ARPE-19 cells from acrolein-induced oxidative damage and mitochondrial dysfunction. A short-term pre-treatment with HTS (48 h) required > 75 μmol/L for showing protection while a long-term pre-treatment (7 days) showed protective effect from 5 μmol/L on. The protective effect of HTS in this model was as potent as that of established mitochondria-targeting antioxidant nutrients. These results suggest that HTS is also a mitochondrial-targeting antioxidant nutrient and that dietary administration of HTS may be an effective measure in reducing and or preventing cigarette smoke-induced or age-related retinal pigment epithelial degeneration, such as age-associated macular degeneration.     

Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress

Nanotechnologies promise new means for drug delivery. ICAM-1 is a good target for vascular immunotargeting of nanoparticles to the perturbed endothelium, although endothelial cells do not internalize monomeric anti-ICAM-1 antibodies. However, coupling ICAM-1 antibodies to nanoparticles creates multivalent ligands that enter cells via an amiloride-sensitive endocytic pathway that does not require clathrin or caveolin. Fluorescence microscopy revealed that internalized anti-ICAM nanoparticles are retained in a stable form in early endosomes for an unusually long time (1-2 h) and subsequently were degraded following slow transport to lysosomes. Inhibition of lysosome acidification by chloroquine delayed degradation without affecting anti-ICAM trafficking. Also, the microtubule disrupting agent nocodazole delayed degradation by inhibiting anti-ICAM nanoparticle trafficking to lysosomes. Addition of catalase to create anti-ICAM nanoparticles with antioxidant activity did not affect the mechanisms of nanoparticle uptake or trafficking. Intracellular anti-ICAM/catalase nanoparticles were active, because endothelial cells were resistant to H2O2-induced oxidative injury for 1-2 h after nanoparticle uptake. Chloroquine and nocodazole increased the duration of antioxidant protection by decreasing the extent of anti-ICAM/catalase degradation. Therefore, the unique trafficking pathway followed by internalized anti-ICAM nanoparticles seems well suited for targeted delivery of therapeutic enzymes to endothelial cells and may provide a basis for treatment of acute vascular oxidative stress.