Interaction of Sirt3 with OGG1 contributes to repair of mitochondrial DNA and protects from apoptotic cell death under oxidative stress

Sirtuin 3 (Sirt3), a major mitochondrial NAD⁺-dependent deacetylase, targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response. In this study, we identified the human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged genome, as a new target protein for Sirt3. We found that Sirt3 physically associated with OGG1 and deacetylated this DNA glycosylase and that deacetylation by Sirt3 prevented the degradation of the OGG1 protein and controlled its incision activity. We further showed that regulation of the acetylation and turnover of OGG1 by Sirt3 played a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress. We observed that following ionizing radiation, human tumor cells with silencing of Sirt3 expression exhibited deteriorated oxidative damage of mtDNA, as measured by the accumulation of 8-oxoG and 4977 common deletion, and showed more severe mitochondrial dysfunction and underwent greater apoptosis in comparison with the cells without silencing of Sirt3 expression. The results reported here not only reveal a new function and mechanism for Sirt3 in defending the mitochondrial genome against oxidative damage and protecting from the genotoxic stress-induced apoptotic cell death but also provide evidence supporting a new mtDNA repair pathway.     

Characterization of the Role of β-Carotene 9,10-Dioxygenase in Macular Pigment Metabolism

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation.     

Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1α-JNK and mitochondrial apoptotic pathway

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H(2)O(2)) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H(2)O(2)-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H(2)O(2)-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H(2)O(2), and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca(2+)) chelator, overexpression of Homer 1a had no significant effects on H(2)O(2)-induced oxidative stress. These results suggest that Homer 1a has protective effects against H(2)O(2)-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca(2+) homeostasis.     

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.     

p62/SQSTM1 is required for the protection against endoplasmic reticulum stress-induced apoptotic cell death

Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding or calcium homeostasis in the ER. To cope with these stresses, ER stress activates the unfolded protein response (UPR) pathway, but unresolved ER stress induces reactive oxygen species (ROS) accumulation leading to apoptotic cell death. However, the mechanisms that underlie protection from ER stress-induced cell death are not clearly defined. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway plays a crucial role in the protection of cells against ROS-mediated oxidative damage. Keap1 acts as a negative regulator of Nrf2 activation. In this study, we investigated the role of the Nrf2-Keap1 pathway in protection from ER stress-induced cell death using tunicamycin (TM) as an ER stress inducer. We found that Nrf2 is an essential protein for the prevention from TM-induced apoptotic cell death and its activation is driven by autophagic Keap1 degradation. Furthermore, ablation of p62, an adapter protein in the autophagy process, attenuates the Keap1 degradation and Nrf2 activation that was induced by TM treatment, and thereby increases susceptibility to apoptotic cell death. Conversely, reinforcement of p62 alleviated TM-induced cell death in p62-deficient cells. Taken together, these results demonstrate that p62 plays an important role in protecting cells from TM-induced cell death through Nrf2 activation.     

Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress

The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax-/- Bak-/- mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.     

Overexpression of mitochondrial methionine sulfoxide reductase B2 protects leukemia cells from oxidative stress-induced cell death and protein damage

According to the mitochondrial theory of aging, mitochondrial dysfunction increases intracellular reactive oxidative species production, leading to the oxidation of macromolecules and ultimately to cell death. In this study, we investigated the role of the mitochondrial methionine sulfoxide reductase B2 in the protection against oxidative stress. We report, for the first time, that overexpression of methionine sulfoxide reductase B2 in mitochondria of acute T-lymphoblastic leukemia MOLT-4 cell line, in which methionine sulfoxide reductase A is missing, markedly protects against hydrogen peroxide-induced oxidative stress by scavenging reactive oxygen species. The addition of hydrogen peroxide provoked a time-gradual increase of intracellular reactive oxygen species, leading to a loss in mitochondrial membrane potential and to protein carbonyl accumulation, whereas in methionine sulfoxide reductase B2-overexpressing cells, intracellular reactive oxygen species and protein oxidation remained low with the mitochondrial membrane potential highly maintained. Moreover, in these cells, delayed apoptosis was shown by a decrease in the cleavage of the apoptotic marker poly(ADP-ribose) polymerase-1 and by the lower percentage of Annexin-V-positive cells in the late and early apoptotic stages. We also provide evidence for the protective mechanism of methionine sulfoxide reductase B2 against protein oxidative damages. Our results emphasize that upon oxidative stress, the overexpression of methionine sulfoxide reductase B2 leads to the preservation of mitochondrial integrity by decreasing the intracellular reactive oxygen species build-up through its scavenging role, hence contributing to cell survival and protein maintenance.     

Early mitochondrial dysfunction in long-lived Mclk1+/- mice

Reduced activity of CLK-1/MCLK1 (also known as COQ7), a mitochondrial enzyme that is necessary for ubiquinone biosynthesis, prolongs the lifespan of nematodes and mice by a mechanism that is distinct from that of the insulin signaling pathway. Here we show that 2-fold reduction of MCLK1 expression in mice reveals an additional function for the protein, as this level of reduction does not affect ubiquinone levels yet affects mitochondrial function substantially. Indeed, we observe that the phenotype of young Mclk1(+/-) mutants includes a severe reduction of mitochondrial electron transport, ATP synthesis, and total nicotinamide adenine dinucleotide (NAD(tot)) pool size as well as an alteration in the activity of key enzymes of the tricarboxylic acid cycle. Surprisingly, we also find that Mclk1 heterozygosity leads to a dramatic increase in mitochondrial oxidative stress by a variety of measures. Furthermore, we find that the mitochondrial dysfunction is accompanied by a decrease in oxidative damage to cytosolic proteins as well as by a decrease in plasma isoprostanes, a systemic biomarker of oxidative stress and aging. We propose a mechanism for the conjunction of low ATP levels, high mitochondrial oxidative stress, and low non-mitochondrial oxidative damage in a long-lived mutant. Our model helps to clarify the relationship between energy metabolism and the aging process and suggests the need for a reformulation of the mitochondrial oxidative stress theory of aging.     

Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, chlorophyceae)

The unicellular green alga Haematococcus pluvialis Flotow is known for its massive accumulation of ketocarotenoids under various stress conditions. Therefore, this microalga is one of the favored organisms for biotechnological production of these antioxidative compounds. Astaxanthin makes up the main part of the secondary carotenoids and is accumulated mostly in an esterified form in extraplastidic lipid vesicles. We have studied phytoene desaturase, an early enzyme of the carotenoid biosynthetic pathway. The increase in the phytoene desaturase protein levels that occurs following induction is accompanied by a corresponding increase of its mRNA during the accumulation period, indicating that phytoene desaturase is regulated at the mRNA level. We also investigated the localization of the enzyme by western-blot analysis of cell fractions and by immunogold labeling of ultrathin sections for electron microscopy. In spite of the fact that secondary carotenoids accumulate outside the chloroplast, no extra pathway specific for secondary carotenoid biosynthesis in H. pluvialis was found, at least at this early stage in the biosynthesis. A transport process of carotenoids from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmatic located lipid vesicles) is implicated.     

Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.     

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1^enu/+ mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1^enu/+ mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.     

Bim links ER stress and apoptosis in cells expressing mutant SOD1 associated with amyotrophic lateral sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.     

Is carotenoid ornamentation linked to the inner mitochondria membrane potential? A hypothesis for the maintenance of signal honesty

Several mechanistic hypotheses have been proposed for how carotenoid pigmentation of integumentary structures can serve as an honest signal of individual quality. These hypotheses are founded on proposed links between carotenoids, immuno responsiveness, and oxidative stress, but an absence of biochemical information on the oxidative pathways of carotenoids has limited the sophistication of such hypotheses. Based on published evidence, we propose that the oxidation of carotenoids for the purpose of ornamentation in birds and reptiles is coupled to the inner mitochondria membrane. We predict that several carotenoid oxidation reactions yielding ornamental pigments occur on the inner mitochondrial membrane. Three of these reactions are proposed to occur within the ubiquinone biosynthesis cluster known as the Coq cluster consisting of approximately a dozen Coq members, tightly integrated and intimately associated with Complex I and III of the electron transport system. Ubiquinone and highly oxidized ornamental carotenoids share a stereochemically-conserved binding region suggesting that these two molecules may have shared similar pathways in the past. Carotenoids and ubiquinones may cooperate as redox participants in anti-radical reactions or independently in helping to maintain membrane or supra-complex stabilization during times of high-energy demand. Under this hypothesis, oxidation of carotenoids is coupled to the inner mitochondria membrane potential such that ornamental coloration reflects the efficiency of cellular respiration.     

Evolution of mitochondrial cell death pathway: Proapoptotic role of HtrA2/Omi in Drosophila

Despite the essential role of mitochondria in a variety of mammalian cell death processes, the involvement of mitochondrial pathway in Drosophila cell death has remained unclear. To address this, we cloned and characterized DmHtrA2, a Drosophila homolog of a mitochondrial serine protease HtrA2/Omi. We show that DmHtrA2 normally resides in mitochondria and is up-regulated by UV-irradiation. Upon receipt of apoptotic stimuli, DmHtrA2 is translocated to extramitochondrial compartment; however, unlike its mammalian counterpart, the extramitochondrial DmHtrA2 does not diffuse throughout the cytosol but stays near the mitochondria. RNAi-mediated knock-down of DmHtrA2 in larvae or adult flies results in a resistance to stress stimuli. DmHtrA2 specifically cleaves Drosophila inhibitor-of-apoptosis protein 1 (DIAP1), a cellular caspase inhibitor, and induces cell death both in vitro and in vivo as potent as other fly cell death proteins. Our observations suggest that DmHtrA2 promotes cell death through a cleavage of DIAP1 in the vicinity of mitochondria, which may represent a prototype of mitochondrial cell death pathway in evolution.     

Oxidative stress is not an obligate mediator of disease provoked by mitochondrial DNA mutations

With age, mitochondrial DNA mutations and oxidative stress increase, leading to the hypothesis that the production of reactive oxygen species causes the pathogenic effects of mitochondrial DNA mutations. We tested this hypothesis using transgenic mice that develop cardiomyopathy due to the accumulation of mitochondrial DNA mutations specifically in the heart. Surprisingly, the mechanism of pathogenesis does not involve increased oxidative stress. The amounts of DNA and protein oxidative adducts are not elevated in the transgenic heart. Neither are signs of increased oxidative stress detected by measurements of enzyme function or oxidative defense systems. Rather, we find that the mitochondrial DNA mutations induce a cytoprotective response including increases in the levels of Bcl-2 and Bfl-1, pro-survival proteins that inhibit apoptosis, and atrial natriuretic factor. Bcl-2 is elevated in nearly all cardiomyocytes before the onset of dilated cardiomyopathy. These results raise the possibility that a signaling pathway between the mitochondrion and the nucleus mediates the pathogenic effect of mitochondrial DNA mutations.     

Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation.

Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.     

Relationship among carotenoid production,copper bioremediation and oxidative stress in Rhodotorula mucilaginosa RCL-11

Rhodotorula mucilaginosa RCL-11, a pigmented yeast isolated from a filter plant of a copper mine in the province of Tucumán, Argentina, supports high concentrations of the heavy metal Cu(II). Copper overload augmented carotenoid biosynthesis in this yeast, modifying at the same time the relative proportion of the pigments produced. Inhibition of the synthesis pathway with diphenylamine suggests an inverse relationship between carotenoid and copper biosorption by R. mucilaginosa RCL-11. The increased activity of superoxide dismutase and catalase measured under inhibition of carotenoid biosynthesis could explain these observations. Exposure to H₂O₂, a second oxidative stress agent, alone or in combination with Cu(II) also modified the carotenoid content, both qualitatively and quantitatively. The change in the relative proportion of the carotenoids torularhodin, torulene and beta-carotena, as well as the detection of gamma-carotene in the presence of H₂O₂ and Cu(II) allows to hypothesize that the carotenoids produced by R. mucilaginosa RCL-11 plays different roles in the oxidative stress response of this yeast.     

Mitochondrial Modulation by Epigallocatechin 3-Gallate Ameliorates Cisplatin Induced Renal Injury through Decreasing Oxidative/Nitrative Stress,Inflammation and NF-kB in Mice

Cancer chemotherapy drug cisplatin is known for its nephrotoxicity. The aim of this study is to investigate whether Epigallocatechin 3-Gallate (EGCG) can reduce cisplatin mediated side effect in kidney and to understand its mechanism of protection against tissue injury. We used a well-established 3-day cisplatin induced nephrotoxicity mice model where EGCG were administered. EGCG is a major active compound in Green Tea and have strong anti-oxidant and anti-inflammatory properties. EGCG protected against cisplatin induced renal dysfunction as measured by serum creatinine and blood urea nitrogen (BUN). EGCG improved cisplatin induced kidney structural damages such as tubular dilatation, cast formation, granulovaculoar degeneration and tubular cell necrosis as evident by PAS staining. Cisplatin induced kidney specific mitochondrial oxidative stress, impaired activities of mitochondrial electron transport chain enzyme complexes, impaired anti-oxidant defense enzyme activities such as glutathione peroxidase (GPX) and manganese superoxide dismutase (MnSOD) in mitochondria, inflammation (tumor necrosis factor α and interleukin 1β), increased accumulation of NF-κB in nuclear fraction, p53 induction, and apoptotic cell death (caspase 3 activity and DNA fragmentation). Treatment of mice with EGCG markedly attenuated cisplatin induced mitochondrial oxidative/nitrative stress, mitochondrial damages to electron transport chain activities and antioxidant defense enzyme activities in mitochondria. These mitochondrial modulations by EGCG led to protection mechanism against cisplatin induced inflammation and apoptotic cell death in mice kidney. As a result, EGCG improved renal function in cisplatin mediated kidney damage. In addition to that, EGCG attenuated cisplatin induced apoptotic cell death and mitochondrial reactive oxygen species (ROS) generation in human kidney tubular cell line HK-2. Thus, our data suggest that EGCG may represent new promising adjunct candidate for cisplatin.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.