Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae. Biochim Biophys Acta

CNR-ISTM Institute of Molecular Science and Technologies, National Council of Research, via Golgi 19, 20133 Milan, Italy.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 12/2010; 1803(12):1376-85. DOI: 10.1016/j.bbamcr.2010.07.007
Source: PubMed


A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells (≈40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).

Download full-text


Available from: Silvia Bradamante
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We here report the apoptotic death of a fungus, Cryptococcus neoformans (C. neoformans), in response to adherence of the pathogenic bacterium Staphylococcus aureus (S. aureus). In co-culture, cryptococcal actin was visibly aggregated. To investigate the mechanism of death, the participation of small GTP(guanosine triphosphate)-binding proteins belonging to the Rho subfamily, which regulate the actin cytoskeleton, was explored. C. neoformans was cultured with S. aureus in the presence of N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide (Y-27632), an inhibitor of Rho-associated coiled-coil forming kinase (ROCK), a downstream effector of Rho. Death of C. neoformans was significantly reduced by the inhibitor. Concomitantly, Y-27632 prevented the aggregation of actin. Therefore, it was concluded that the Rho/ROCK pathway is involved in cell death induced by adherence stress. Increased expression of the voltage-dependent anion channel (VDAC), located in the mitochondrial outer membrane, has previously been observed in the apoptosis-like death of C. neoformans in the presence of hydrogen peroxide. Ruthenium red (RuR), which binds to VDAC and inhibits cytochrome c release, was used to determine the involvement of VDAC following adherence stress caused by S. aureus. RuR treatment increased the viability of C. neoformans co-cultured with S. aureus in a dose dependent manner. These findings suggest that Rho-ROCK signaling could be involved, via a mitochondrial pathway, in the apoptosis-like death of C. neoformans induced by the adherence of S. aureus.
    Preview · Article · Jun 2011 · Microbiology and Immunology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Actin is one of the most abundant and highly conserved proteins of all eukaryotes. It is the major component of the cytoskeleton, and is involved in many of the structural and dynamic aspects of cell growth, differentiation, division, membrane organisation, transport, and signal transduction. Alterations in such a critical component can lead to pathological conditions. We here describe the effects of actin cytoskeleton disorganisation and/or depolymerisation in the Saccharomyces cerevisiae yeast model system. The structure of the actin cytoskeleton was disorganised by subjecting yeast cells to simulated (Rotating Wall Vessel) or real microgravity (spaceflight), both of which activated the signal transduction cascade of the high osmolarity glycerol (HOG) MAP kinase pathway, which responds to cell swelling/shrinking, and the cell wall integrity (CWI) pathway, which is involved in cell wall biogenesis and actin cytoskeleton reorganisation. The same results were observed when the actin cytoskeleton structure was depolymerised by means of treatment with dihydrocytochalasin B (DHCB) or (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632). The HOG and CWI activation indicate a response to a variation in cell volume. Under such conditions, yeast activates volume-sensitive ion channels that alter the ion flux to restore normal volume. These alterations are not pathological per se but, in the case of significant environmental stress (such as oxidative stress), they can lead to clear signs of damage. We observed massive protein carbonylation and a marked loss of the antioxidant glutathione through chloride channels. These findings suggest interrelationships between the actin cytoskeleton, cell volume regulation and the loss of antioxidant defences, and provide new insights into the underlying cause of the GSH depletion associated with many human diseases, such as cancer, neurodegenerative disorders, and HIV- and aging-related diseases.
    No preview · Article · Jan 2012
  • [Show abstract] [Hide abstract]
    ABSTRACT: Treatment with molecular hydrogen alleviates microgravity-induced bone loss through abating oxidative stress, restoring osteoblastic differentiation, and suppressing osteoclast differentiation and osteoclastogenesis. INTRODUCTION: Recently, it has been suggested that hydrogen gas exerts a therapeutic antioxidant activity by selectively reducing cytotoxic reactive oxygen species (ROS). The aim of the present study was to elucidate whether treatment with molecular hydrogen alleviated bone loss induced by modeled microgravity in rats. METHODS: Hindlimb suspension (HLS) and rotary wall vessel bioreactor were used to model microgravity in vivo and in vitro, respectively. Sprague-Dawley rats were exposed to HLS for 6 weeks to induced bone loss and simultaneously administrated with hydrogen water (HW). Then, we investigated the effects of incubation with hydrogen-rich medium (HRM) on MC3T3-E1 and RAW264.7 cells exposed to modeled microgravity. RESULTS: Treatment with HW alleviated HLS-induced reduction of bone mineral density, ultimate load, stiffness, and energy in femur and lumbar vertebra. Treatment with HW alleviated HLS-induced augmentation of malondialdehyde content and peroxynitrite content and reduction of total sulfhydryl content in femur and lumbar vertebra. In cultured MC3T3-E1 cells, incubation with HRM inhibited modeled microgravity-induced ROS formation, reduction of osteoblastic differentiation, increase of ratio of receptor activator of nuclear factor kappa B ligand to osteoprotegerin, inducible nitric oxide synthetase upregulation, and Erk1/2 phosphorylation. In cultured RAW264.7, incubation with HRM aggravated modeled microgravity-induced ROS formation, osteoclastic differentiation, and osteoclastogenesis. CONCLUSION: Treatment with molecular hydrogen alleviates microgravity-induced bone loss in rats. Molecular hydrogen could thus be envisaged as a nutritional countermeasure for spaceflight but remains to be tested in humans.
    No preview · Article · May 2012 · Osteoporosis International
Show more