Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis

Ferritin protein nanocages are the main iron store in mammals. They have been predicted to fulfil the same function in plants but direct evidence was lacking. To address this, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. Loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron, as shown by reduced growth and strong defects in flower development. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporters genes in the stalk, over-accumulation of iron in reproductive organs, and a decrease in fertility. Finally, we show that, in the absence of ferritin, plants have higher levels of reactive oxygen species, and increased activity of enzymes involved in their detoxification. Seed germination also showed higher sensitivity to pro-oxidant treatments. Arabidopsis ferritins are therefore essential to protect cells against oxidative damage.     

Agrobacterium tumefaciens fur has important physiological roles in iron and manganese homeostasis, the oxidative stress response, and full virulence

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.     

Effect of iron supplementation on oxidative stress and antioxidant status in iron-deficiency anemia

This study was designed to measure the effect of iron supplementation on antioxidant status in iron-deficient anemia, including the time for hemoglobin normalization and at the time of filling of iron body stores. The extent of plasma lipid peroxidation was evaluated by measuring the levels of malondialdehyde and glutathione peroxidase (GSH-Px), and the activities of superoxide dismutase (SOD) and catalase in 63 patients with iron-deficiency anemia before and after 6 wk of iron supplementation and at the time when body iron stores are saturated. After 6 wk of iron supplementation, a significant decrease of oxidative stress was observed in the treated subjects relative to controls (p<0.05). No significant differences existed between treated patients at 6 wk and at the end of the study. The erythrocyte levels of catalase, SOD, and GSH-Px were significantly lower in treated patients relative to controls (p<0.05). These levels increased after 6 wk of supplementation (p<0.05) and showed no significant differences with those at the end of the study.     

The role of extracellular vesicles in iron homeostasis and ferroptosis: Focus on musculoskeletal diseases

Iron homeostasis is crucial for maintaining proper cellular function, and its disruption is considered one of the pathogenic mechanisms underlying musculoskeletal diseases. Under conditions of oxidative stress, the accumulation of cellular iron overload and lipid peroxidation can lead to ferroptosis. Extracellular vesicles (EVs), serving as mediators in the cell-to-cell communication, play an important role in regulating the outcome of cell ferroptosis. Growing evidence has proven that EV biogenesis and secretion are tightly associated with cellular iron export. Furthermore, different sources of EVs deliver diverse cargoes to bring about phenotypic changes in the recipient cells, either activating or inhibiting ferroptosis. Thus, delivering therapies targeting ferroptosis through EVs may hold significant potential for treating musculoskeletal diseases. This review aims to summarize current knowledge on the role of EVs in iron homeostasis and ferroptosis, as well as their therapeutic applications in musculoskeletal diseases, and thereby provide valuable insights for both research and clinical practice.     

miR-210 over-expression enhances mesenchymal stem cell survival in an oxidative stress environment through antioxidation and c-Met pathway activation

microRNA-210(miR-210)has generally been reported to be associated with cell survival under hypoxia.However,there are few data regarding the role of miR-210 in the survival of mesenchymal stem cells(MSCs)under oxidative stress conditions.Thus,we sought to investigate whether miR-210 over-expression could protect MSCs against oxidative stress injury and what the primary mechanisms involved are.The results showed that over-expression of miR-210 significantly reduced the apoptosis of MSCs under oxidative stress,accompanied by obvious increases in cell viability and superoxide dismutase activity and remarkable decreases in malonaldehyde content and reactive oxygen species production,resulting in a noticeable reduction of apoptotic indices when compared with the control.Moreover,the above beneficial effects of miR-210 could be significantly reduced by c-Met pathway repression.Collectively,these results showed that miR-210 over-expression improved MSC survival under oxidative stress through antioxidation and c-Met pathway activation,indicating the potential development of a novel approach to enhance the efficacy of MSC-based therapy for injured myocardium.     

Three proline rotamases involved in calcium homeostasis play differential roles in stress tolerance,virulence and calcineurin regulation of Beauveria bassiana

FK506‐sensitive proline rotamases (FPRs), also known as FK506‐binding proteins (FKBPs), can mediate immunosuppressive drug resistance in budding yeast but their physiological roles in filamentous fungi remain opaque. Here, we report that three FPRs (cytosolic/nuclear 12.15‐kD Fpr1, membrane‐associated 14.78‐kD Fpr2 and nuclear 50.43‐kD Fpr3) are all equally essential for cellular Ca²⁺ homeostasis and contribute significantly to calcineurin activity at different levels in the insect‐pathogenic fungus Beauveria bassiana although the deletion of fpr1 alone conferred resistance to FK506. Radial growth, conidiation, conidial viability and virulence were less compromised in the absence of fpr1 or fpr2 than in the absence of fpr3, which abolished almost all growth on scant media and reduced growth moderately on rich media. The Δfpr3 mutant was more sensitive to Na⁺, K⁺, Mn²⁺, Ca²⁺, Cu²⁺, metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn²⁺, Mg²⁺, Fe²⁺, H₂O₂ and cell wall‐perturbing agents. In contrast, the Δfpr1 mutant was less sensitive to fewer stress cues. Most of 32 examined genes involved in DNA damage repair, Na⁺/K⁺ detoxification or osmotolerance and Ca²⁺ homeostasis were downregulated sharply in Δfpr2 and Δfpr3 but rarely so affected in Δfpr1, coinciding well with their phenotypic changes. These findings uncover important, but differential, roles of three FPRs in the fungal adaptation to insect host and environment and provide novel insight into their essential roles in calcium signalling pathway.     

Activation of Nrf2/Keap1 signaling and autophagy induction against oxidative stress in heart in iron deficiency

We investigated the effects of dietary iron deficiency on the redox system in the heart. Dietary iron deficiency increased heart weight and accumulation of carbonylated proteins. However, expression levels of heme oxygenase-1 and LC3-II, an antioxidant enzyme and an autophagic marker, respectively, in iron-deficient mice were upregulated compared to the control group, resulting in a surrogate phenomenon against oxidative stress.     

Effect of iron addition on mAb productivity and oxidative stress in Chinese hamster ovary culture

Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro-oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria-generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron-induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.     

Dietary vitamin C and E modulates oxidative stress induced-kidney and lens injury in diabetic aged male rats through modulating glucose homeostasis and antioxidant systems

Diabetes induces oxidative stress in aged human and rat, although daily supplementation of vitamins C and E (VCE) can be beneficial to aged diabetic rats by reducing free radical production. The aim of the present study was to evaluate whether dietary VCE supplementation relieves oxidative stress in streptozotocin (STZ)-induced diabetic in aged rats. Thirty aged rats were randomly divided into three groups. The first group was used as a control. The second group was made diabetic using a single dose of intraperitoneal STZ. VCE-supplemented feed was given to aged diabetic rats constituting the third group. On the 21st day of the experiment, blood, lens and kidney samples were taken from all animals. Glutathione peroxidase (GSH-Px) activity in lens and kidney, reduced glutathione (GSH), vitamin E and β-carotene concentrations in kidney were lower in the diabetic group than in the control whereas plasma glucose, urea and creatinine, and kidney and lens peroxidation (LP) levels were higher in the diabetic group than in the control. However, kidney and lens LP levels, and plasma glucose, urea and creatinine values were decreased by VCE supplementation. Lens and kidney GSH-Px activity, kidney GSH, vitamin E and β-carotene concentrations and erythrocyte counts were increased by VCE treatment. Kidney weights, vitamin A, haemoglobin, hematocrit, leukocyte and platelets values were not changed by diabetes and/or VCE supplementation. VCE ameliorated also diabetes-induced histopathological changes in kidney. In conclusion, we observed that VCE supplementation is beneficial towards kidney and lens of aged diabetic rats by modulating oxidative and antioxidant systems.     

Effects of an autocrine factor deficit on the survival and energy metabolism of CTLL-2 cells under oxidative stress conditions in culture

An increasing body of data shows that survival of mammalian cells is under the control of growth factors and autocrine survival factors (AF). We studied the effects of an AF deficit on the survival, intracellular ATP content and the transmembrane potential of mitochondria in IL-2-dependent CTLL-2 cells following oxidative stress. We show that cells cultivated under conditions of AF deficit became more susceptible to oxidative injury in comparison with CTLL-2 cells cultivated without an AF deficit (the control cells); their death occurred at lower H₂O₂ concentrations than in the case of control cells. The ATP content in CTLL-2 cells decreased under an AF deficit even without stress; treatment of these cells with hydrogen peroxide led to an additional significant decrease of ATP content, which was accompanied by injury of the cell membrane (blebbing) and by a sharp fall in mitochondrial potential. Cell death after oxidative stress under conditions of AF deficit was shown to proceed along both the apoptosis and necrosis pathways.     

Two proteins from Saccharomyces cerevisiae: Pfy1 and Pkc1, play a dual role in activating actin polymerization and in increasing cell viability in the adaptive response to oxidative stress

In this work, we show that the proteins Pkc1 and Pfy1 play a role in the repolarization of the actin cytoskeleton and in cell survival in response to oxidative stress. We have also developed an assay to determine the actin polymerization capacity of total protein extracts using fluorescence recovery after photobleaching techniques and actin purified from rabbit muscle. This assay allowed us to demonstrate that Pfy1 promotes actin polymerization under conditions of oxidative stress, while Pkc1 induces actin polymerization and cell survival under all the conditions tested. Our assay also points to a relationship between Pkc1 and Pfy1 in the actin cytoskeleton polymerization that is required to adapt to oxidative stress.     

Deuterated docosahexaenoic acid protects against oxidative stress and geographic atrophy‐like retinal degeneration in a mouse model with iron overload

Oxidative stress plays a central role in age‐related macular degeneration (AMD). Iron, a potent generator of hydroxyl radicals through the Fenton reaction, has been implicated in AMD. One easily oxidized molecule is docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in photoreceptor membranes. Oxidation of DHA produces toxic oxidation products including carboxyethylpyrrole (CEP) adducts, which are increased in the retinas of AMD patients. In this study, we hypothesized that deuterium substitution on the bis‐allylic sites of DHA in photoreceptor membranes could prevent iron‐induced retinal degeneration by inhibiting oxidative stress and lipid peroxidation. Mice were fed with either DHA deuterated at the oxidation‐prone positions (D‐DHA) or control natural DHA and then given an intravitreal injection of iron or control saline. Orally administered D‐DHA caused a dose‐dependent increase in D‐DHA levels in the neural retina and retinal pigment epithelium (RPE) as measured by mass spectrometry. At 1 week after iron injection, D‐DHA provided nearly complete protection against iron‐induced retinal autofluorescence and retinal degeneration, as determined by in vivo imaging, electroretinography, and histology. Iron injection resulted in carboxyethylpyrrole conjugate immunoreactivity in photoreceptors and RPE in mice fed with natural DHA but not D‐DHA. Quantitative PCR results were consistent with iron‐induced oxidative stress, inflammation, and retinal cell death in mice fed with natural DHA but not D‐DHA. Taken together, our findings suggest that DHA oxidation is central to the pathogenesis of iron‐induced retinal degeneration. They also provide preclinical evidence that dosing with D‐DHA could be a viable therapeutic strategy for retinal diseases involving oxidative stress.     

Autophagy and ER stress play an essential role in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol

Chronic lymphocytic leukemia (CLL) is a mature B cell malignancy and is the most prevalent type of leukemia in adults. There is no curative therapy for this disease; however, several new agents have shown very promising results. Autophagy has not been studied in CLL and in this study we first sought to determine if autophagy was functional in CLL with classic inducers, and if this contributes to direct cytotoxicity or protection from cell death. While autophagy is activated with all classic stimuli of this process, only unfolded protein endoplasmic reticulum (ER) stress-mediated autophagy protects from cell death. Interestingly, select therapeutic agents (fludarabine, GS-1101, flavopiridol), which are active in CLL, also induce autophagy. Of interest, only the broad cyclin-dependent kinase inhibitor flavopiridol has improved efficacy when autophagy is antagonized biochemically (chloroquine) or by siRNA. This promoted an investigation which demonstrated unexpectedly that flavopiridol mediates ER stress and downstream activation of MAP3K5/ASK1, which ultimately is responsible for cell death. Similarly, autophagy activated in part via ER stress and also CDK5 inhibition is protective against cell death induced by this process. Collectively, our studies demonstrate that in CLL, autophagy is induced by multiple stimuli but only acts as a mechanism of resistance against ER stress-mediating agents. Similarly, flavopiridol mediates ER stress as a primary mechanism of action in CLL, and autophagy serves as a mechanism of resistance to this agent.     

DJ‐1 plays an obligatory role in the cardioprotection of delayed hypoxic preconditioning against hypoxia/reoxygenation‐induced oxidative stress through maintaining mitochondrial complex I activity

DJ‐1 was recently reported to mediate the cardioprotection of delayed hypoxic preconditioning (DHP) by suppressing hypoxia/reoxygenation (H/R)‐induced oxidative stress, but its mechanism against H/R‐induced oxidative stress during DHP is not fully elucidated. Here, using the well‐established cellular model of DHP, we again found that DHP significantly improved cell viability and reduced lactate dehydrogenase release with concurrently up‐regulated DJ‐1 protein expression in H9c2 cells subjected to H/R. Importantly, DHP efficiently improved mitochondrial complex I activity following H/R and attenuated H/R‐induced mitochondrial reactive oxygen species (ROS) generation and subsequent oxidative stress, as demonstrated by a much smaller decrease in reduced glutathione/oxidized glutathione ratio and a much smaller increase in intracellular ROS and malondialdehyde contents than that observed for the H/R group. However, the aforementioned effects of DHP were antagonized by DJ‐1 knockdown with short hairpin RNA but mimicked by DJ‐1 overexpression. Intriguingly, pharmacological inhibition of mitochondria complex I with Rotenone attenuated all the protective effects caused by DHP and DJ‐1 overexpression, including maintenance of mitochondria complex I and suppression of mitochondrial ROS generation and subsequent oxidative stress. Taken together, this work revealed that preserving mitochondrial complex I activity and subsequently inhibiting mitochondrial ROS generation could be a novel mechanism by which DJ‐1 mediates the cardioprotection of DHP against H/R‐induced oxidative stress damage.     

Towards Alzheimer's root cause: ECSIT as an integrating hub between oxidative stress, inflammation and mitochondrial dysfunction. Hypothetical role of the adapter protein ECSIT in familial and sporadic Alzheimer's disease pathogenesis

Here we postulate that the adapter protein evolutionarily conserved signalling intermediate in Toll pathway (ECSIT) might act as a molecular sensor in the pathogenesis of Alzheimer's disease (AD). Based on the analysis of our AD-associated protein interaction network, ECSIT emerges as an integrating signalling hub that ascertains cell homeostasis by the specific activation of protective molecular mechanisms in response to signals of amyloid-beta or oxidative damage. This converges into a complex cascade of patho-physiological processes. A failure to repair would generate severe mitochondrial damage and ultimately activate pro-apoptotic mechanisms, promoting synaptic dysfunction and neuronal death. Further support for our hypothesis is provided by increasing evidence of mitochondrial dysfunction in the disease etiology. Our model integrates seemingly controversial hypotheses for familial and sporadic forms of AD and envisions ECSIT as a biomarker to guide future therapies to halt or prevent AD.