Cytoprotective effects of 12-oxo phytodienoic acid,a plant-derived oxylipin jasmonate,on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells

BackgroundJasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity.MethodsThe cells were pretreated with individual jasmonates for 24 h and exposed to hydrogen peroxide (H₂O₂) for 24 h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA).ResultsAmong the jasmonates, only OPDA suppressed H₂O₂-induced cytotoxicity. OPDA pretreatment also inhibited the H₂O₂-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2.ConclusionsThese results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway.General significancePlant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.     

H2O2 preferentially synergizes with nitroprusside to induce apoptosis associated with superoxide dismutase dysregulation in human melanoma irrespective of p53 status: Antagonism by o-phenanthroline

The pro-oxidant hydrogen peroxide (H₂O₂) is converted to a reactive oxygen species by transition metals like iron. Since mutations in the p53 tumor suppressor gene contribute to drug resistance, we used genetically-matched human C8161 melanoma harbouring wt or DN-R175H mutant p53, to investigate the influence of p53 status on the potentiation of H₂O₂ toxicity by: (a) intact sodium nitroprusside or nitroferricyanide (SNP), (b) its light-exhausted NO-depleted form (lex-SNP), (c) potassium ferricyanide, or (d) ferric ammonium sulphate. Whereas single treatments with SNP or H₂O₂ were partly cytotoxic, preferentially potentiation of H₂O₂ toxicity was evidenced with intact or lex-SNP. No comparable increase of H₂O₂ toxicity was induced by ferricyanide, ferric ammonium sulphate or S-nitroso-N-acetyl penicillamine (SNAP), a known NO donor lacking iron. Immune blotting revealed apoptosis-associated PARP cleavage induced by [SNP + H₂O₂] irrespective of p53 status. This correlated with an eightfold induction of [Mn-SOD; SOD2] in wt p53 melanoma cells, and with a super-induction of the same enzyme reciprocal with loss of [Cu,Zn-SOD; SOD1], in mutant p53 cells. All these changes were antagonized by the anti-oxidant N-acetylcysteine or the iron chelator o-phenanthroline. We hypothesize that superoxide dismutase imbalance and iron-dependent redox changes involving OH species generated from a Fenton reaction between [SNP + H₂O₂], may be important in this anti-tumor activity. Although tumor drug resistance is frequently associated with DN-p53 mutations, our data shows for the first time the preferential ability of SNP to enhance H₂O₂ toxicity, irrespective of p53 status.     

Enhancement of PPAR-β activity by repetitive low-grade H2O2 stress protects human umbilical vein endothelial cells from subsequent oxidative stress-induced apoptosis

Repetitive stress has been shown to up-regulate antioxidant defense and increase survival after subsequent oxidative injury. The up-regulation of antioxidant defense has been identified as an underlying cause of the apoptosis-inhibitory effects exerted by repetitive stress. However, it remains unclear what the important signaling mechanisms are by which cells preexposed to low-grade stress deal with apoptosis-inducing stress. In this study, we repetitively stressed human umbilical vein endothelial cells (HUVECs) through multiple exposures to a low dose (30 μM) of H₂O₂ in culture for 4 weeks. We then examined the effects of repetitive stress on PPAR-β expression and activity as well as the role of PPAR-β in the protective potency of repetitive stress. Our results show that repetitive stress enhances PPAR-β expression and activity, thereby inhibiting oxidative stress-induced apoptosis. Further, PPAR-β-directed antisense oligonucleotides reduced the PPAR-β protein content, enhanced the H₂O₂-mediated apoptosis, and ablated the protective effect of repetitive low-grade H₂O₂ stress. The specific PPAR-β agonist L-165041 significantly potentiated the apoptosis induced by H₂O₂ (p < 0.05) and increased the protective effect of repetitive stress. These findings indicate that repetitive low-grade H₂O₂ stress protects HUVECs from subsequent oxidative stress-induced apoptosis by enhancing PPAR-β expression and activity.     

Phytochemical screening of flavonoids with their antioxidant activities from rapeseed (Brassica napus L.)

Ten flavone compounds, including three new flavonoid glycosides, were isolated from defatted rapeseed, and their protective antioxidant effect on H₂O₂-induced oxidative damage in human umbilical vein endothelial cells (ECV-304) was investigated. Three new flavonoid glycosides were identified as kaempferol-3-O-[(6-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (8), kaempferol-3,7-di-O-β-d-glucopyranoside-4'-O-(6-O-sinapoyl)-β-d-glucopyranoside (9), and kaempferol-3-O-[(3-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (10). The protective effects of all of the isolated compounds on H₂O₂-induced oxidative damage were assessed, and the activities of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured. All of compounds had a protective effect on H₂O₂-induced oxidative damage in ECV-304 cells and the presence of a substituted sinapoyl group and its position in the structures were used to elucidate the activity differences.     

Role of intracellular labile iron,ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide

To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H₂O₂-resistant Jurkat T cell line “HJ16” was developed by gradual adaptation of parental “J16” cells to high concentrations of H₂O₂. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H₂O₂-induced oxidative damage and necrotic cell death (up to 3 mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H₂O₂-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H₂O₂ concentrations higher than 0.1 mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H₂O₂ concentrations higher than 0.1 mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H₂O₂ treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H₂O₂ treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H₂O₂ treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H₂O₂ has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to oxidant-mediated glutathione depletion, FtH modulation, and labile iron release and a significant increase in FtMt levels following H₂O₂ treatment.     

Modulation of antioxidant enzyme activities and metabolites ratios by nitric oxide in short-term salt stressed soybean root nodules

Several abiotic factors cause molecular damage to plants either directly or through the accumulation of reactive oxygen species such as hydrogen peroxide (H₂O₂). We investigated if application of nitric oxide (NO) donor 2,2′-(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) could reduce the toxic effect resulting from short-term salt stress. Salt treatment (150 mM NaCl) alone and in combination with 10 μM DETA/NO or 10 μM DETA were given to matured soybean root nodules for 24 h. Salt stress resulted in high H₂O₂ level and lipid peroxidation while application of DETA/NO effectively reduced H₂O₂ level and prevented lipid peroxidation in the soybean root nodules. NO treatment increased the activities of ascorbate peroxidase and dehydroascorbate reductase under salt stress. Whereas short-term salt stress reduced AsA/DHAsA and GSH/GSSG ratios, application of the NO donor resulted in an increase of the reduced form of the antioxidant metabolites thus increasing the AsA/DHAsA and GSH/GSSG ratios. Our data suggests a protective role of NO against salt stress.     

SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-κB

Oxidative stress-mediated cell death in cardiomyocytes reportedly plays an important role in many cardiac pathologies. Our previous report demonstrated that mitochondrial SIRT3 plays an essential role in mediating cell survival in cardiac myocytes, and that resveratrol protects cardiomyocytes from oxidative stress-induced apoptosis by activating SIRT3. However, the exact mechanism by which SIRT3 prevents oxidative stress remains unknown. Here, we show that exposure of H9c2 cells to 50 μM H₂O₂ for 6 h caused a significant increase in cell death and the down-regulation of SIRT3. Reactive oxygen species (ROS)-mediated NF-κB activation was involved in this SIRT3 down-regulation. The SIRT3 activator, resveratrol, which is considered an important antioxidant, protected against H₂O₂-induced cell death, whereas the SIRT inhibitor, nicotinamide, enhanced cell death. Moreover, resveratrol negatively regulated H₂O₂-induced NF-κB activation, whereas nicotinamide enhanced H₂O₂-induced NF-κB activation. We also found that SOD2, Bcl-2 and Bax, the downstream genes of NF-κB, were involved in this pathological process. These results suggest that SIRT3 protects cardiomyocytes exposed to oxidative stress from apoptosis via a mechanism that may involve the NF-κB pathway.     

Genetic structure of expanding wolf (Canis lupus) populations in Italy and Croatia,and the early steps of the recolonization of the Eastern Alps

After centuries of range contraction and demographic declines wolves are now expanding in Europe, colonizing regions from where they have been absent for centuries. Wolf colonizing the western Alps originate by the expansion of the Italian population. Vagrant wolves of Italian and Dinaric-Balkan origins have been recently observed in the Eastern Alps. In this study we compared the genetic structure of wolf populations in Italy and Croatia, aiming to identify the sources of the ongoing recolonization of the Eastern Alps. DNA samples, extracted from 282 Italian and 152 Croatian wolves, were genotyped at 12 autosomal microsatellites (STR), four Y-linked STR and at the hypervariable part of the mitochondrial DNA control-region (mtDNA CR1). Wolves in Croatia and Italy underwent recent demographic bottlenecks, but they differ in genetic diversity and population structure. Wolves in Croatia were more variable at STR loci (N_A = 7.4, H_O = 0.66, H_E = 0.72; n = 152) than wolves in Italy (N_A = 5.3, H_O = 0.57, H_E = 0.58; n = 282). We found four mitochondrial DNA (mtDNA CR1) and 11 Y-STR haplotypes in Croatian wolves, but only one mtDNA CR1 and three Y-STR haplotypes in Italy. Wolves in Croatia were subdivided into three genetically distinct subpopulations (in Dalmatia, Gorski kotar and Lika regions), while Italian wolves were not sub-structured. Assignment testing shows that the eastern and central Alps are recolonized by wolves dispersing from both the Italian and Dinaric populations. The recolonization of the Alps will predictably continue in the future and the new population will be genetically admixed and very variable with greater opportunities for local adaptations and survival.     

Antioxidant cytoprotection by peroxisomal peroxiredoxin-5

Peroxiredoxin-5 (PRDX5) is a thioredoxin peroxidase that reduces hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This enzyme is present in the cytosol, mitochondria, peroxisomes, and nucleus in human cells. Antioxidant cytoprotective functions have been previously documented for cytosolic, mitochondrial, and nuclear mammalian PRDX5. However, the exact function of PRDX5 in peroxisomes is still not clear. The aim of this work was to determine the function of peroxisomal PRDX5 in mammalian cells and, more specifically, in glial cells. To study the role of PRDX5 in peroxisomes, the endogenous expression of PRDX5 in murine oligodendrocyte 158 N cells was silenced by RNA interference. In addition, human PRDX5 was also overexpressed in peroxisomes using a vector coding for human PRDX5, whose unconventional peroxisomal targeting sequence 1 (PTS1; SQL) was replaced by the prototypical PTS1 SKL. Stable 158 N clones were obtained. The antioxidant cytoprotective function of peroxisomal PRDX5 against peroxisomal and mitochondrial KillerRed-mediated reactive oxygen species production as well as H₂O₂ was examined using MTT viability assays, roGFP2, and C11-BOBIPY probes. Altogether our results show that peroxisomal PRDX5 protects 158 N oligodendrocytes against peroxisomal and mitochondrial KillerRed- and H₂O₂-induced oxidative stress.     

Increased proton leak and SOD2 expression in myotubes from obese non-diabetic subjects with a family history of type 2 diabetes

Muscle insulin resistance is linked to oxidative stress and decreased mitochondrial function. However, the exact cause of muscle insulin resistance is still unknown. Since offspring of patients with type 2 diabetes mellitus (T2DM) are susceptible to developing insulin resistance, they are ideal for studying the early development of insulin resistance. By using primary muscle cells derived from obese non-diabetic subjects with (FH +) or without (FH ?) a family history of T2DM, we aimed to better understand the link between mitochondrial function, oxidative stress, and muscle insulin resistance. Insulin-stimulated glucose uptake and glycogen synthesis were normal in FH + myotubes. Resting oxygen consumption rate was not different between groups. However, proton leak was higher in FH + myotubes. This was associated with lower ATP content and decreased mitochondrial membrane potential in FH + myotubes. Surprisingly, mtDNA content was higher in FH + myotubes. Oxidative stress level was not different between FH + and FH ? groups. Reactive oxygen species content was lower in FH + myotubes when differentiated in high glucose/insulin (25 mM/150 pM), which could be due to higher oxidative stress defenses (SOD2 expression and uncoupled respiration). The increased antioxidant defenses and mtDNA content in FH + myotubes suggest the existence of compensatory mechanisms, which may provisionally prevent the development of insulin resistance.     

Effect of acetone extract from stem bark of Acacia species (A. dealbata,A. ferruginea and A. leucophloea) on antioxidant enzymes status in hydrogen peroxide-induced HepG2 cells

Acacia species are multipurpose trees, widely used in the traditional systems of medicine to treat various ailments. The major objective of the present study was to determine the gene expression of enzymatic antioxidants by acetone extract from the stem bark of three Acacia species (Acacia dealbata, Acacia ferruginea and Acacia leucophloea) in hydrogen peroxide (H₂O₂)-induced human hepatoma (HepG2) cells. The expression of antioxidant enzymes such as superoxide dismutase containing copper–zinc (CuZnSOD)/manganese (MnSOD), catalase (CAT) and glutathione peroxidase (GPx) in HepG2 cells was evaluated by real-time PCR. The results of antioxidant enzyme expression in real-time PCR study revealed that the H₂O₂ (200 μM) challenged HepG2 cells reduced the expression of enzymes such as SOD, GPx and CAT. However, the cells pre-treated with acetone extracts of all the three Acacia species significantly (P > 0.05) up-regulated the expression of antioxidant enzymes in a concentration dependent manner (25, 50 and 75 μg/mL). In conclusion, the findings of our study demonstrated that the acetone extract of Acacia species effectively inhibited H₂O₂ mediated oxidative stress and may be useful as a therapeutic agent in preventing oxidative stress mediated diseases.     

Exendin-4 protects adipose-derived mesenchymal stem cells from apoptosis induced by hydrogen peroxide through the PI3K/Akt–Sfrp2 pathways

Adipose-derived mesenchymal stem cells (ADMSCs)-based therapy is a promising modality for the treatment of myocardial infarction in the future. However, the majority of transplanted cells are readily lost after transplantation because of hypoxia and oxidative stress. An efficient means to enhance the ability of ADMSCs to survive under pathologic conditions is required. In our study, we explored the effects of exendin-4 (Ex-4) on ADMSCs apoptosis in vitro induced by hydrogen peroxide, focusing in particular on mitochondrial apoptotic pathways and PI3K/Akt–secreted frizzled-related protein 2 (Sfrp2) survival signaling. We demonstrated that ADMSCs subjected to H₂O₂ for 12 h exhibited impaired mitochondrial function and higher apoptotic rate. However, Ex-4 (1–20 nM) preconditioning for 12 h could protect ADMSCs against H₂O₂-mediated apoptosis in a dose-dependent manner. Furthermore, Ex-4 pretreatment upregulated the levels of superoxide dismutase and glutathione as well as downregulating the production of reactive oxygen species and malondialdehyde. Western blots revealed that increased antiapoptotic proteins Bcl-2 and c-IAP1/2 as well as decreased proapoptotic proteins Bax and cytochrome c appeared in ADMSCs with Ex-4 incubation, which inhibited the caspase-9-involved mitochondrial apoptosis pathways with evidence showing inactivation of caspase-9/3 and preservation of mitochondrial membrane potential. Furthermore, we illustrated that Ex-4 enhanced Akt phosphorylation, which increased the expression of Sfrp2. Notably, blockade of the PI3K/Akt pathway or knockdown of Sfrp2 with siRNA obviously abolished the protective effects of Ex-4 on mitochondrial function and ADMSCs apoptosis under H₂O₂. In summary, this study confirmed that H₂O₂ induced ADMSCs apoptosis through mitochondria-dependent cell death pathways, and Ex-4 preconditioning may reduce such apoptosis of ADMSCs through the PI3K/Akt–Sfrp2 pathways.     

Protective effects of aloe-emodin on scopolamine-induced memory impairment in mice and H2O2-induced cytotoxicity in PC12 cells

Aloe-emodin (AE) is one of the most important active components of Rheum officinale Baill. The present study aimed to investigate that AE could attenuate scopolamine-induced cognitive deficits via inhibiting acetylcholinesterase (AChE) activity and modulating oxidative stress. Kunming (KM) mice were received intraperitoneal injection of scopolamine (2 mg/kg) to induce cognitive impairment. Learning and memory performance were assessed in the Morris water maze (MWM). After behavioral testing, the mice were sacrificed and their hippocampi were removed for biochemical assays (superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), AChE and acetylcholine (ACh)). In vitro, we also performed the AChE activity assay and H₂O₂-induced PC12 cells toxicity assay. After 2 h exposure to 200 μM H₂O₂ in PC12 cells, the cytotoxicity were evaluated by cell viability (MTT), nitric oxide (NO)/lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) production. Our results confirmed that AE showed significant improvement in cognitive deficit in scopolamine-induced amnesia animal model. Besides, it increased SOD, GPx activities and ACh content, while decreased the level of MDA and AChE activity in AE treated mice. In addition, AE was found to inhibit AChE activity (IC₅₀ = 18.37 μg/ml) in a dose-dependent manner. Furthermore, preincubation of PC12 cells with AE could prevent cytotoxicity induced by H₂O₂ and reduce significantly extracellular release of NO, LDH and intracellular accumulation of ROS. The study indicated that AE could have neuroprotective effects against Alzheimer’s disease (AD) via inhibiting the activity of AChE and modulating oxidative stress.     

Design,synthesis and evaluation of novel indandione derivatives as multifunctional agents with cholinesterase inhibition,anti-β-amyloid aggregation,antioxidant and neuroprotection properties against Alzheimer’s disease

A series of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)-1H-indene-1,3(2H)-diones were designed, synthesized and appraised as multifunctional anti-Alzheimer agents. In vitro studies of compounds 27–38 showed that these compounds exhibit moderate to excellent AChE, BuChE and Aβ aggregation inhibitory activity. Notably, compounds 34 and 38 appeared as most active multifunctional agents in the entire series and exhibited excellent inhibition against AChE (IC₅₀ = 0.048 μM: 34; 0.036 μM: 38), Aβ aggregation (max% inhibition 82.2%, IC₅₀ = 9.2 μM: 34; max% inhibition 80.9%, IC₅₀ = 10.11 μM: 38) and displayed significant antioxidant potential in ORAC-FL assay. Both compounds also successfully diminished H₂O₂ induced oxidative stress in SH-SY5Y cells. Fascinatingly, compounds 34 and 38 showed admirable neuroprotective effects against H₂O₂ and Aβ induced toxicity in SH-SY5Y cells. Additionally, both derivatives showed no considerable toxicity in neuronal cell viability assay and represented drug likeness properties in the primarily pharmacokinetics study. All these results together, propelled out that compounds 34 and 38 might serve as promising multi-functional lead candidates for treatment of AD in the future.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Elicitation with abiotic stresses improves pro-health constituents,antioxidant potential and nutritional quality of lentil sprouts

Phenolic content and antioxidant potential of lentil sprouts may be enhanced by treatment of seedlings in abiotic stress conditions without any negative influence on nutritional quality.The health-relevant and nutritional quality of sprouts was improved by elicitation of 2-day-old sprouts with oxidative, osmotic, ion-osmotic and temperature stresses. Among the sprouts studied, those obtained by elicitation with osmotic (600 mM mannitol) and ion-osmotic (300 mM NaCl) shocks had the highest total phenolic content levels: 6.52 and 6.56 mg/g flour, respectively. Oxidative stress significantly enhanced the levels of (+)-catechin and p-coumaric acid. A marked elevation of the chlorogenic and gallic acid contents was also determined for sprouts induced at 4 °C and 40 °C. The elevated phenolic content was translated into the antioxidant potential of sprouts, especially the ability to reduce lipid oxidation. A marked elevation of this ability was determined for seedlings treated with 20 mM, 200 mM H₂O₂ (oxidative stress) and 600 mM mannitol (osmotic stress); about a 12-fold, 8-fold and 9.5-fold increase in respect to control sprouts. The highest ability to quench free radicals was observed in sprouts induced by osmotic stress (IC₅₀- 4.91 and 5.12 mg/ml for 200 mM and 600 mM mannitol, respectively). The highest total antioxidant activity indexes were determined for sprouts elicited with 20 mM H₂O₂ and 600 mM mannitol: 4.0 and 3.4, respectively. All studied growth conditions, except induction at 40 °C, caused a significant elevation of resistant starch levels which was also affected in a subsequent reduction of starch digestibility.Improvement of sprout quality by elicitation with abiotic stresses is a cheap and easy biotechnology and it seems to be an alternative to conventional techniques applied to improve the health promoting phytochemical levels and bioactivity of low-processed food.     

Regulation of mitochondrial FoF1ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977 bp deletion of mitochondrial DNA

Sirt3, a mitochondrial NAD⁺-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F_oF₁ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F_oF₁ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F_oF₁ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F_oF₁ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F_oF₁ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ? 85% of mtDNA with 4977 bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F_oF₁ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5–10 μM of menadione impaired the Sirt3-mediated deacetylation and activation on F_oF₁ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F_oF₁ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.