Increased Superoxide Levels Research Articles

Biosensor-Based Assessment of Pesticides and Mineral Fertilizers’ Influence on Ecotoxicological Parameters of Soils under Soya, Sunflower and Wheat

Pesticides and fertilizers used in agriculture can negatively affect the soil, increasing its toxicity. In this work, a battery of whole-cell bacterial lux-biosensors based on the E. coli MG1655 strain with various inducible promoters, as well as the natural luminous Vibrio aquamarinus VKPM B-11245 strain, were used to assess the effects of agrochemical soil treatments. The advantages of using biosensors are sensitivity, specificity, low cost of analysis, and the ability to assess the total effect of toxicants on a living cell and the type of their toxic effect. Using the V. aquamarinus VKPM B-11245 strain, the synergistic effect of combined soil treatment with pesticides and mineral fertilizers was shown, which led to an increase in the overall (integral) toxicity of soils higher than that of the individual application of substances. Several probable implementation mechanisms of agrochemical toxic effects have been discovered. DNA damage caused by both SOS response induction and alkylation, oxidative stress due to increased superoxide levels, and damage to cellular proteins and membranes are among them. Thus, the usage of biosensors makes it possible to assess the cumulative effect of various toxicants on living organisms without using expensive chemical analyses.

MUC1-C integrates aerobic glycolysis with suppression of oxidative phosphorylation in triple-negative breast cancer stem cells

Activation of the MUC1-C protein promotes lineage plasticity, epigenetic reprogramming, and the cancer stem cell (CSC) state. The present studies performed on enriched populations of triple-negative breast cancer (TNBC) CSCs demonstrate that MUC1-C is essential for integrating activation of glycolytic pathway genes with self-renewal and tumorigenicity. MUC1-C further integrates the glycolytic pathway with suppression of mitochondrial DNA (mtDNA) genes encoding components of mitochondrial Complexes I-V. The repression of mtDNA genes is explained by MUC1-C-mediated (i)downregulation of the mitochondrial transcription factor A (TFAM) required for mtDNA transcription and (ii) induction of the mitochondrial transcription termination factor 3 (mTERF3). In support of pathogenesis that suppresses mitochondrial ROS production, targeting MUC1-C increases (i)mtDNA gene transcription, (ii) superoxide levels, and (iii) loss of self-renewal capacity. These findings and scRNA-seq analysis of CSC subpopulations indicate that MUC1-C regulates self-renewal and redox balance by integrating activation of glycolysis with suppression of oxidative phosphorylation.

Everolimus prevents doxorubicin-induced apoptosis in H9c2 cardiomyocytes but not in MCF-7 cancer cells: Cardioprotective roles of autophagy, mitophagy, and AKT

Cardiotoxicity is a severe side effect of the chemotherapeutic agent doxorubicin (DOX). We recently showed that DOX-induced cardiomyocyte apoptosis and death were attenuated through autophagy pre-induction. Herein, we assessed how the autophagy/mitophagy-inducing antitumor drug everolimus (EVL) affected DOX-induced cytotoxicity in the rat cardiomyocyte cell line H9c2 and human breast cancer cell line MCF-7. Apoptosis was assessed using annexin V assay. Autophagy and mitophagy were assessed using fluorescence assays. Cellular protein levels were determined using western blotting. Pretreatment with EVL (1 nM) before DOX exposure inhibited mammalian target of rapamycin (mTOR) activity, induced autophagy and mitophagy, and activated protein kinase B (AKT) in H9c2 cells. In mitochondria, DOX (1 μM) induced structural damage (decreased membrane potential and release of cytochrome c), increased superoxide levels, decreased apoptosis inhibitor Bcl-2, and increased apoptosis inducer Bax, leading to apoptosis and reduced viability in H9c2 cells. EVL pretreatment suppressed DOX-induced changes. EVL anti-apoptotic effects were inhibited by treatment with MK-2206, a selective AKT inhibitor. Furthermore, EVL suppressed DOX-induced cardiotoxicity through autophagy/mitophagy and AKT activation but did not attenuate DOX-induced apoptosis or reduction in viability in MCF-7 cells. Altogether, EVL can protect cardiomyocytes from DOX-induced apoptosis and toxicity without reducing DOX antitumor effects, allowing safer chemotherapy.

NECAB2 is an endosomal protein important for striatal function

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

A New Quinone-Based Inhibitor of Mitochondrial Complex I in D-Conformation, Producing Invasion Reduction and Sensitization to Venetoclax in Breast Cancer Cells.

Mitochondrial Complex I plays a crucial role in the proliferation, chemoresistance, and metastasis of breast cancer (BC) cells. This highlights it as an attractive target for anti-cancer drugs. Using submitochondrial particles, we identified FRV-1, an ortho-carbonyl quinone, which inhibits NADH:duroquinone activity in D-active conformation and reduces the 3ADP state respiration dependent on Complex I, causing mitochondrial depolarization, ATP drop, increased superoxide levels, and metabolic remodeling towards glycolysis in BC cells. Introducing methyl groups at FRV-1 structure produced analogs that acted as electron acceptors at the Complex I level or increased the inhibitory effect of FCCP-stimulated oxygen consumption rate, which correlated with their redox potential, but increased toxicity on RMF-621 human breast fibroblasts was observed. FRV-1 was inactive in the naphthoquinone oxidoreductase 1 (NOQ1)-positive BC cell line, MCF7, but the sensitivity was recovered by dicoumarol, a NOQ1 inhibitor, suggesting that FRV-1 is a NOQ1 substrate. Importantly, FRV-1 selectively inhibited the proliferation, migration, and invasion of NQO1 negative BC cell, MDA-MB-231, in an OXPHOS- and ROS-dependent manner and sensitized it to the BH3 mimetic drug venetoclax. Overall, FRV-1 is a novel Complex I inhibitor in D-active conformation, blocking possibly the re-activation to A-state, producing selective anti-cancer effects in NQO1-negative BC cell lines.

5-Hydroxymethylfurfural reduces skeletal muscle superoxide production and modifies force production in rats exposed to hypobaric hypoxia.

Decreased blood-tissue oxygenation at high altitude (HA) increases mitochondrial oxidant production and reduces exercise capacity. 5-Hydroxymethylfurfural (5-HMF) is an antioxidant that increases hemoglobin's binding affinity for oxygen. For these reasons, we hypothesized that 5-HMF would improve muscle performance in rats exposed to a simulated HA of ~5500 m. A secondary objective was to measure mitochondrial activity and dynamic regulation of fission and fusion because they are linked processes impacted by HA. Fisher 344 rats received 5-HMF (40 mg/kg/day) or vehicle during exposure to sea level or HA for 72 h. Right ankle plantarflexor muscle function was measured pre- and post-exposure. Post-exposure measurements included arterial blood gas and complete blood count, flexor digitorum brevis myofiber superoxide production and mitochondrial membrane potential (ΔΨm), and mitochondrial dynamic regulation in the soleus muscle. HA reduced blood oxygenation, increased superoxide levels and lowered ΔΨm, responses that were accompanied by decreased peak isometric torque and force production at frequencies >75 Hz. 5-HMF increased isometric force production and lowered oxidant production at sea level. In HA exposed animals, 5-HMF prevented a decline in isometric force production at 75-125 Hz, prevented an increase in superoxide levels, further decreased ΔΨm, and increased mitochondrial fusion 2 protein expression. These results suggest that 5-HMF may prevent a decrease in hypoxic force production during submaximal isometric contractions by an antioxidant mechanism.

Exposure to Stress Alters Cardiac Gene Expression and Exacerbates Myocardial Ischemic Injury in the Female Murine Heart.

Mental stress is a risk factor for myocardial infarction in women. The central hypothesis of this study is that restraint stress induces sex-specific changes in gene expression in the heart, which leads to an intensified response to ischemia/reperfusion injury due to the development of a pro-oxidative environment in female hearts. We challenged male and female C57BL/6 mice in a restraint stress model to mimic the effects of mental stress. Exposure to restraint stress led to sex differences in the expression of genes involved in cardiac hypertrophy, inflammation, and iron-dependent cell death (ferroptosis). Among those genes, we identified tumor protein p53 and cyclin-dependent kinase inhibitor 1A (p21), which have established controversial roles in ferroptosis. The exacerbated response to I/R injury in restraint-stressed females correlated with downregulation of p53 and nuclear factor erythroid 2-related factor 2 (Nrf2, a master regulator of the antioxidant response system-ARE). S-female hearts also showed increased superoxide levels, lipid peroxidation, and prostaglandin-endoperoxide synthase 2 (Ptgs2) expression (a hallmark of ferroptosis) compared with those of their male counterparts. Our study is the first to test the sex-specific impact of restraint stress on the heart in the setting of I/R and its outcome.

Clinical efficacy and safety of two different hematoporphyrin monomethyl ether-mediated photodynamic therapy regimen in Chinese children with port-wine stain.

Hematoporphyrin monomethyl ether-photodynamic therapy (HMME-PDT) has achieved encouraging clinical outcomes in adult port-wine stain (PWS). Optimal treatment option for children with PWS was minimal. To compare whether the clinical effectiveness of HMME-PDT with the 5-min (fast) administration treatment regimen (FATR) was better than the 20-min (slow) administration treatment regimen (SATR) for PWS of children in vivo and in vitro. Thirty-four children with PWS were divided into two groups including FATR and SATR. The two groups received three times HMME-PDT, respectively. Treatment efficacy and safety were evaluated in vivo and in vitro. Erythema index (EI) was used to evaluate the clinical outcomes. Both FATR and SATR were effective and safe in children with PWS after HMME-PDT. There were significance differences between the two groups in reductions of EI after the second treatment (p < 0.001) and the third treatment (p < 0.001) with HMME-PDT. The serum HMME concentration reach the peak level at short time compare with SATR group. A significance increased superoxide levels were observed in FATR group compare to SATR groups in vitro (p < 0.05). Our study suggested that HMME-PDT was effective and safe for children with PWS, the therapy regimen with FATR was better in clinical efficacy than that of the SATR.

Morphological and mitochondrial changes in murine choroid plexus epithelial cells during healthy aging

BackgroundChoroid plexuses (ChPs) are intraventricular structures mainly composed by specialized epithelial cells interconnected by tight junctions that establish the blood-cerebrospinal fluid (CSF) barrier. ChPs are essential to produce CSF and transport solutes from and into the brain. Deterioration of ChP function and morphology has been correlated to worsening of neurodegenerative disorders. We here map morpho-functional changes in the ChP epithelial cells during healthy aging, starting from young adult to 2-years old mice.MethodsWe used a multi-tiered approach, including transmission electron microscopy (TEM), immunohistochemistry, RT-qPCR, Western Blot and 2-photon microscopy (2-PM) at multiple timepoints ranging from young adult to 2-years old mice.ResultsWe identified distinct morpho-functional modifications in epithelial cells of ChP starting from 8 to 12 months of age, which mostly remained stable up to 2 years. These changes include flattening of the epithelium, reduction of microvilli length and an augmentation of interrupted tight junctions. We also found a decrease in mitochondria density together with elongation of mitochondria in older mice. Morphological mitochondrial rearrangements were accompanied by increased superoxide levels, decreased membrane potential and decreased mitochondrial motility in aged mice. Interestingly, most of the age-related changes were not accompanied by modification of protein and/or gene expression levels and aged mitochondria effectively responded to acute pharmacological stressful stimuli.ConclusionsOur study suggests a long-term progression of multiple morpho-functional features of the mouse choroid plexus epithelium during adulthood followed by structural remodeling during the aging process. These findings can lead to a better understanding on how functional and morphological rearrangements of ChP are correlated during aging.

Analysis of Electric Field Stimulation in Blue Light Stressed 661W Cells

Though electrical stimulation is used as a therapeutic approach to treat retinal and spinal injuries, many protective mechanisms at cellular level have not been elucidated. We performed a detailed analysis of cellular events in blue light (Li) stressed 661W cells, which were subjected to direct current electric field (EF) stimulation. Our findings revealed that EF stimulation induced protective effects in 661W cells from Li-induced stress by multiple defense mechanisms, such as increase in mitochondrial activity, gain in mitochondrial potential, increase in superoxide levels, and the activation of unfolded protein response (UPR) pathways, all leading to an enhanced cell viability and decreased DNA damage. Here, our genetic screen results revealed the UPR pathway to be a promising target to ameliorate Li-induced stress by EF stimulation. Thus, our study is important for a knowledgeable transfer of EF stimulation into clinical application.

GAT107-mediated α7 nicotinic acetylcholine receptor signaling attenuates inflammatory lung injury and mortality in a mouse model of ventilator-associated pneumonia by alleviating macrophage mitochondrial oxidative stress via reducing MnSOD-S-glutathionylation

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial and viral infections, causing ventilator-associated pneumonia (VAP). Compromised host defense and inflammatory lung injury are mediated, in part, by high extracellular concentrations of HMGB1, which can be decreased by GTS-21, a partial agonist of α7 nicotinic acetylcholine receptor (α7nAChR). Here, we report that a novel α7nAChR agonistic positive allosteric modulator (ago-PAM), GAT107, at 3.3 mg/kg, i.p., significantly decreased animal mortality and markers of inflammatory injury in mice exposed to hyperoxia and subsequently infected with Pseudomonas aeruginosa. The incubation of macrophages with 3.3 μM of GAT107 significantly decreased hyperoxia-induced extracellular HMGB1 accumulation and HMGB1-induced macrophage phagocytic dysfunction. Hyperoxia-compromised macrophage function was correlated with impaired mitochondrial membrane integrity, increased superoxide levels, and decreased manganese superoxide dismutase (MnSOD) activity. This compromised MnSOD activity is due to a significant increase in its level of glutathionylation. The incubation of hyperoxic macrophages with 3.3 μM of GAT107 significantly decreases the levels of glutathionylated MnSOD, and restores MnSOD activity and mitochondrial membrane integrity. Thus, GAT107 restored hyperoxia-compromised phagocytic functions by decreasing HMGB1 release, most likely via a mitochondrial-directed pathway. Overall, our results suggest that GAT107 may be a potential treatment to decrease acute inflammatory lung injury by increasing host defense in patients with VAP.

Endothelial Extracellular Signal-Regulated Kinase/Thromboxane A2/Prostanoid Receptor Pathway Aggravates Endothelial Dysfunction and Insulin Resistance in a Mouse Model of Metabolic Syndrome.

Background Metabolic syndrome is characterized by insulin resistance, which impairs intracellular signaling pathways and endothelial NO bioactivity, leading to cardiovascular complications. Extracellular signal-regulated kinase (ERK) is a major component of insulin signaling cascades that can be activated by many vasoactive peptides, hormones, and cytokines that are elevated in metabolic syndrome. The aim of this study was to clarify the role of endothelial ERK2 in vivo on NO bioactivity and insulin resistance in a mouse model of metabolic syndrome. Methods and Results Control and endothelial-specific ERK2 knockout mice were fed a high-fat/high-sucrose diet (HFHSD) for 24 weeks. Systolic blood pressure, endothelial function, and glucose metabolism were investigated. Systolic blood pressure was lowered with increased NO products and decreased thromboxane A2/prostanoid (TP) products in HFHSD-fed ERK2 knockout mice, and Nω-nitro-l-arginine methyl ester (L-NAME) increased it to the levels observed in HFHSD-fed controls. Acetylcholine-induced relaxation of aortic rings was increased, and aortic superoxide level was lowered in HFHSD-fed ERK2 knockout mice. S18886, an antagonist of the TP receptor, improved endothelial function and decreased superoxide level only in the rings from HFHSD-fed controls. Glucose intolerance and the impaired insulin sensitivity were blunted in HFHSD-fed ERK2 knockout mice without changes in body weight. In vivo, S18886 improved endothelial dysfunction, systolic blood pressure, fasting serum glucose and insulin levels, and suppressed nonalcoholic fatty liver disease scores only in HFHSD-fed controls. Conclusions Endothelial ERK2 increased superoxide level and decreased NO bioactivity, resulting in the deterioration of endothelial function, insulin resistance, and steatohepatitis, which were improved by a TP receptor antagonist, in a mouse model of metabolic syndrome.

Dipeptide Repeat Pathology in C9orf72-ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance.

The hexanucleotide expansion of the C9orf72 gene is found in 40% of familial amyotrophic lateral sclerosis (ALS) patients. This genetic alteration has been connected with impaired management of reactive oxygen species. In this study, we conducted targeted transcriptional profiling in leukocytes from C9orf72 patients and control subjects by examining the mRNA levels of 84 redox-related genes. The expression of ten redox genes was altered in samples from C9orf72 ALS patients compared to healthy controls. Considering that Nuclear factor erythroid 2-Related Factor 2 (NRF2) modulates the expression of a wide range of redox genes, we further investigated its status on an in vitro model of dipeptide repeat (DPR) toxicity. This model mimics the gain of function, toxic mechanisms attributed to C9orf72 pathology. We found that exposure to DPRs increased superoxide levels and reduced mitochondrial potential as well as cell survival. Importantly, cells overexpressing DPRs exhibited reduced protein levels of NRF2 and its target genes upon inhibition of the proteasome or its canonical repressor, the E3 ligase adapter KEAP1. However, NRF2 activation was sufficient to recover cell viability and redox homeostasis. This study identifies NRF2 as a putative target in precision medicine for the therapy of ALS patients harboring C9orf72 expansion repeats.

Diabetes Aggravates Photoreceptor Pathologies in a Mouse Model for Ocular Vitamin A Deficiency.

Emerging evidence indicates that diabetes disturbs photoreceptor function and vitamin A homeostasis. However, the biochemical basis of this phenotype is not well established. Here, we compared the effects of streptozotocin-induced diabetes in wild-type (WT) mice and Stra6-/- mice, a mouse model for ocular vitamin A deficiency. After 8 weeks, diabetes increased serum retinyl esters in mice of both genotypes. The eyes of diabetic WT mice displayed increased superoxide levels but no changes in retinoid concentrations. Diabetic Stra6-/- mice showed increased ocular retinoid concentrations, but superoxide levels remained unchanged. After 30 weeks, significant alterations in liver and fat retinoid concentrations were observed in diabetic mice. Diabetic WT mice exhibited a decreased expression of visual cycle proteins and a thinning of the photoreceptor layer. Stra6-/- mice displayed significantly lower ocular retinoid concentration than WT mice. An altered retinal morphology and a reduced expression of photoreceptor marker genes paralleled these biochemical changes and were more pronounced in the diabetic animals. Taken together, we observed that diabetes altered vitamin A homeostasis in several organ systems and aggravated photoreceptor pathologies in the vitamin-deficient mouse eyes.

17β-Oestradiol facilitates M2 macrophage skewing and ameliorates arrhythmias in ovariectomized female infarcted rats.

Epidemiological studies have suggested a lower incidence of arrhythmia‐induced sudden cardiac death in women than in men. 17β‐oestradiol (E2) has been reported to have a post‐myocardial infarction antiarrhythmic effect, although the mechanisms have yet to be elucidated. We investigated whether E2‐mediated antioxidation regulates macrophage polarization and affects cardiac sympathetic reinnervation in rats after MI. Ovariectomized Wistar rats were randomly assigned to placebo pellets, E2 treatment, or E2 treatment +3‐morpholinosydnonimine (a peroxynitrite generator) and followed for 4 weeks. The infarct sizes were similar among the infarcted groups. At Day 3 after infarction, post‐infarction was associated with increased superoxide levels, which were inhibited by administering E2. E2 significantly increased myocardial IL‐10 levels and the percentage of regulatory M2 macrophages compared with the ovariectomized infarcted alone group as assessed by immunohistochemical staining, Western blot and RT‐PCR. Nerve growth factor colocalized with both M1 and M2 macrophages at the magnitude significantly higher in M1 compared with M2. At Day 28 after infarction, E2 was associated with attenuated myocardial norepinephrine levels and sympathetic hyperinnervation. These effects of E2 were functionally translated in inhibiting fatal arrhythmias. The beneficial effect of E2 on macrophage polarization and sympathetic hyperinnervation was abolished by 3‐morpholinosydnonimine. Our results indicated that E2 polarized macrophages into the M2 phenotype by inhibiting the superoxide pathway, leading to attenuated nerve growth factor‐induced sympathetic hyperinnervation after myocardial infarction.

Deletion of rifampicin-inactivating mono-ADP-ribosyl transferase gene of Mycobacterium smegmatis globally altered gene expression profile that favoured increase in ROS levels and thereby antibiotic resister generation.

The physiological role of mono-ADP-ribosyl transferase (Arr) of Mycobacterium smegmatis, which inactivates rifampicin, remains unclear. An earlier study reported increased expression of arr during oxidative stress and DNA damage. This suggested a role for Arr in the oxidative status of the cell and its associated effect on DNA damage. Since reactive oxygen species (ROS) influence oxidative status, we investigated whether Arr affected ROS levels in M. smegmatis. Significantly elevated levels of superoxide and hydroxyl radical were found in the mid-log phase (MLP) cultures of the arr knockout strain (arr-KO) as compared those in the wild-type strain (WT). Complementation of arr-KO with expression from genomically integrated arr under its native promoter restored the levels of ROS equivalent to that in WT. Due to the inherently high ROS levels in the actively growing arr-KO, rifampicin resisters with rpoB mutations could be selected at 0 hr of exposure itself against rifampicin, unlike in the WT where the resisters emerged at 12th hr of rifampicin exposure. Microarray analysis of the actively growing cultures of arr-KO revealed significantly high levels of expression of genes from succinate dehydrogenase I and NADH dehydrogenase I operons, which would have contributed to the increased superoxide levels. In parallel, expression of specific DNA repair genes was significantly decreased, favouring retention of the mutations inflicted by the ROS. Expression of several metabolic pathway genes also was significantly altered. These observations revealed that Arr was required for maintaining a gene expression profile that would provide optimum levels of ROS and DNA repair system in the actively growing M. smegmatis.

Endothelial Colony-Forming Cells Dysfunctions Are Associated with Arterial Hypertension in a Rat Model of Intrauterine Growth Restriction.

Infants born after intrauterine growth restriction (IUGR) are at risk of developing arterial hypertension at adulthood. The endothelium plays a major role in the pathogenesis of hypertension. Endothelial colony-forming cells (ECFCs), critical circulating components of the endothelium, are involved in vasculo-and angiogenesis and in endothelium repair. We previously described impaired functionality of ECFCs in cord blood of low-birth-weight newborns. However, whether early ECFC alterations persist thereafter and could be associated with hypertension in individuals born after IUGR remains unknown. A rat model of IUGR was induced by a maternal low-protein diet during gestation versus a control (CTRL) diet. In six-month-old offspring, only IUGR males have increased systolic blood pressure (tail-cuff plethysmography) and microvascular rarefaction (immunofluorescence). ECFCs isolated from bone marrow of IUGR versus CTRL males displayed a decreased proportion of CD31+ versus CD146+ staining on CD45− cells, CD34 expression (flow cytometry, immunofluorescence), reduced proliferation (BrdU incorporation), and an impaired capacity to form capillary-like structures (Matrigel test), associated with an impaired angiogenic profile (immunofluorescence). These dysfunctions were associated with oxidative stress (increased superoxide anion levels (fluorescent dye), decreased superoxide dismutase protein expression, increased DNA damage (immunofluorescence), and stress-induced premature senescence (SIPS; increased beta-galactosidase activity, increased p16INK4a, and decreased sirtuin-1 protein expression). This study demonstrated an impaired functionality of ECFCs at adulthood associated with arterial hypertension in individuals born after IUGR.

Systematic Assessment of Uncoupling in Flavoprotein Oxidases and Monooxygenases

Flavin as a cofactor is an extremely versatile molecule that participates in a wide range of biochemical reactions. A special characteristic of the flavin cofactor, unique for a metal free cofactor, is its ability to react in its reduced form with molecular oxygen. This is exploited in flavoprotein oxidases and monooxygenases. The flavin-mediated reduction of dioxygen can lead to various reactive oxygen species (ROS), primarily hydrogen peroxide and superoxide. No systematic analysis of the formation of such reduced oxygen species produced by flavoprotein oxidases and monooxygenases had been performed before. In this work, we investigated which ROS are formed by several prototype flavoprotein oxidases and monooxygenases: phenylacetone monooxygenase (TfPAMO), an engineered TfPAMO variant that acts as an NADPH oxidase (TfPAMO C65D), eugenol oxidase (RjEUGO), and 5-hydroxymethyl furfural oxidase (MHMFO). The formed amounts of superoxide and hydrogen peroxide were measured under various conditions (different substrate concentrations, pH values and cosolvents). Interestingly, all flavoenzymes were found to produce, except for hydrogen peroxide, significant amounts of superoxide. Moreover, increased superoxide levels were measured at higher pH, which could be indicative for a pH-sensitive caged radical pair dissociation. To probe the effect of ROS formation on biocatalytic performance, conversions catalyzed by TfPAMO or RjEUGO, with or without catalase, were monitored. This revealed that catalase has a beneficial effect. No detrimental effect of the accumulation of superoxide on biocatalysis could be demonstrated. The results reveal that formation of ROS by flavoenzymes is highly dependent on the experimental conditions used. The results provide a better insight into the mechanism by which ROS is formed in flavoenzymes and may help studies or applications in which ROS formation should be promoted or minimized for industrial biocatalysis.

Fucoxanthin induces prostate cancer PC-3 cell apoptosis by causing mitochondria dysfunction and oxidative stress

To investigate the apoptosis- inducing effect of fucoxanthin in human prostate cancer PC-3 cells and the underlying mechanism. The viability and apoptosis of PC-3 cells treated with fucoxanthin were analyzed using commercial kits, and the mitochondrial membrane potential, mitochondrial morphology and mitochondrial superoxide were detected using fluorescence probe staining. The contents of ATP, H2O2, malondialdehyde (MDA), superoxide and the total antioxidant capacity of PC-3 cells were determined. The protein expressions of Bcl-2, Bax and cytochrome c were detected with Western blotting, and the activity of caspase-9 and caspase- 3/7 was detected using corresponding kits. Fucoxanthin significantly inhibited the viability of PC-3 cells in a time- and dose-dependent manner, and dose-dependently induced apoptosis of the cells (P < 0.05). Fucoxanthin-treated PC-3 cells showed significantly decreased mitochondrial membrane potential, mitochondrial fragmentation and increased superoxide level in the mitochondria (P < 0.05), and these effects of fucoxanthin were dose- dependent. Fucoxanthin dose-dependently decreased ATP level and the total antioxidant capacity of PC-3 cells, increased the contents of H2O2, MDA and superoxide (all P < 0.05), enhanced the protein expressions of Bax and cytochrome c in the cytoplasm, and lowered the protein expressions of Bcl-2 and cytochromes in the mitochondria (P < 0.05). Fucoxanthin induces apoptosis of PC-3 cells by triggering mitochondrial dysfunction to cause oxidative stress and by activating mitochondria-mediated apoptotic signaling pathways, suggesting its potential in prostate cancer treatment.

Insight into the Interactome of Intramitochondrial PKA Using Biotinylation-Proximity Labeling.

Mitochondria are fully integrated in cell signaling. Reversible phosphorylation is involved in adjusting mitochondrial physiology to the cellular needs. Protein kinase A (PKA) phosphorylates several substrates present at the external surface of mitochondria to maintain cellular homeostasis. However, few targets of PKA located inside the organelle are known. The aim of this work was to characterize the impact and the interactome of PKA located inside mitochondria. Our results show that the overexpression of intramitochondrial PKA decreases cellular respiration and increases superoxide levels. Using proximity-dependent biotinylation, followed by LC-MS/MS analysis and in silico phospho-site prediction, we identified 21 mitochondrial proteins potentially targeted by PKA. We confirmed the interaction of PKA with TIM44 using coimmunoprecipitation and observed that TIM44-S80 is a key residue for the interaction between the protein and the kinase. These findings provide insights into the interactome of intramitochondrial PKA and suggest new potential mechanisms in the regulation of mitochondrial functions.