Transcription Of Antioxidant Enzymes Research Articles

Sedanolide Activates KEAP1-NRF2 Pathway and Ameliorates Hydrogen Peroxide-Induced Apoptotic Cell Death.

Sedanolide is a bioactive compound with anti-inflammatory and antitumor activities. Although it has been recently suggested that sedanolide activates the nuclear factor E2-related factor 2 (NRF2) pathway, there is little research on its effects on cellular resistance to oxidative stress. The objective of the present study was to investigate the function of sedanolide in suppressing hydrogen peroxide (H2O2)-induced oxidative damage and the underlying molecular mechanisms in human hepatoblastoma cell line HepG2 cells. We found that sedanolide activated the antioxidant response element (ARE)-dependent transcription mediated by the nuclear translocation of NRF2. Pathway enrichment analysis of RNA sequencing data revealed that sedanolide upregulated the transcription of antioxidant enzymes involved in the NRF2 pathway and glutathione metabolism. Then, we further investigated whether sedanolide exerts cytoprotective effects against H2O2-induced cell death. We showed that sedanolide significantly attenuated cytosolic and mitochondrial reactive oxygen species (ROS) generation induced by exposure to H2O2. Furthermore, we demonstrated that pretreatment with sedanolide conferred a significant cytoprotective effect against H2O2-induced cell death probably due to preventing the decrease in the mitochondrial membrane potential and the increase in caspase-3/7 activity. Our study demonstrated that sedanolide enhanced cellular resistance to oxidative damage via the activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 pathway.

Abstract 17696: Perm1 Stabilizes Nrf2 via Keap1 Oxidation to Protect the Heart Against Reperfusion Injury

Introduction: Reperfusion after ischemia (IR) produces reactive oxygen species (ROS) that promote myocardial injury. Nrf2 is a transcription factor that promotes transcription of antioxidant enzymes. Keap1 is a negative regulator of Nrf2. When oxidated by ROS, Keap1 releases Nrf2, leading to Nrf2 activation. Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) is a scaffold or adaptor protein that maintains cellular metabolism. However, whether and how Perm1 protects the heart against IR injury via Nrf2 remains unknown. Methods: We used systemic Perm1 knockout (KO) mice and adeno-associated virus (AAV)-induced cardiac-specific Perm1 overexpression to investigate the pathological role of Perm1 in IR injury. Results: IR-induced myocardial infarction was greater in Perm1KO mice than in control mice. Consistently, infarction was smaller in mice with Perm1 overexpression (Infarction size/area at risk (AAR): AAV-GFP as a control 0.41 and AAV-Perm1 0.28*; p<0.05). Myocardial oxidative stress, evaluated via increases in dityrosine, 4HNE and GSSG/GSH, after IR was greater in Perm1KO but less in Perm1 overexpression mice. Expression of IR-induced Nrf2 target genes, such as catalase, superoxide dismutase 2, heme oxygenase 1, NADH quinone dehydrogenase 1 and thioredoxin 1, was inhibited in Perm1KO but promoted in overexpression mice. The detrimental effects observed in Perm1KO mice were normalized by ML334, an inhibitor of Keap1-Nrf2 interaction (Infarction size/AAR: Wild 0.42; Perm1KO 0.58; Wild with ML334 0.37 and Perm1KO with ML334 0.42*; p<0.05 vs. Perm1KO), suggesting that Perm1 protects the heart via Nrf2. In primary cultured rat cardiomyocytes, Perm1 was bound to Keap1 and promoted its oxidation, thereby stabilizing and activating Nrf2. Perm1-induced Nrf2 activation was verified with reporter gene assays using a consensus binding sequence of Nrf2, termed antioxidant response element. In an in vitro system using recombinant proteins, Perm1 directly bound to Keap1. Conclusions: These results suggest that Perm1 is a novel regulator of cellular redox homeostasis through Nrf2 activation via Keap1 oxidation that protects the heart against IR injury.

Protective Effect of Iris germanica L. Rhizome-Derived Exosome against Oxidative-Stress-Induced Cellular Senescence in Human Epidermal Keratinocytes

Plant-derived exosomes can exert therapeutic effects against various dermatological conditions. Several studies have demonstrated that plant-derived exosomes can have positive effects on the skin, preventing aging, hyperpigmentation, and hair loss. In this study, the protective effects of Iris germanica L. rhizome-derived exosomes (Iris-exosomes) on oxidative-stress-induced cellular dysfunction were investigated in human epidermal keratinocytes (nHEKs). Iris-exosomes with a diameter range of 100–300 nm were detected. In the cytotoxicity assay, Iris-exosomes with up to 107 particles per milliliter were found to possess no cytotoxicity, and we recovered H2O2-induced cell viability loss. In nHEKs, H2O2-induced ROS levels were significantly reduced using Iris-exosomes and additionally associated with increases in antioxidant enzyme transcription. The H2O2-induced SA-β-gal-positive nHEKs were decreased using Iris-exosomes; these effects correlate with the changed levels of cell cycle arrest marker p21. Furthermore, the H2O2-induced loss of in vitro wound-healing properties and early detection of keratin 1 and 10—keratinization markers—were restored to control levels using Iris-exosomes. Altogether, these results indicate the possibility that Iris-exosomes exert antioxidant and anti-senescence effects in order to protect against oxidative-stress-induced cellular dysfunction in nHEKs.

Suppressing plant defense: Scavenge the ROS!

Reactive oxygen species (ROS)-mediated defense against fungal pathogens is an essential arm of plant immunity. As a counter defense, these pathogens synthesize antioxidant enzymes that scavenge the ROS produced by plants. The molecular mechanism behind the upregulation of these enzymes in fungal pathogens was unknown. A recent study by Zhang et al. (2023, https://doi.org/10.15252/embj.2022112634) has shed light on the mechanism, and it has been shown that deacetylation of FolSrpk1 protein on the K304 residue following oxidative stress is an important event in the signaling cascade leading to ROS detoxification in Fusarium oxysporum f. sp. lycopersici. Deacetylated FolSrpk1 moves to the nucleus where it hyperphosphorylates FolSr1, which further regulates the transcription of antioxidant enzymes. This mechanism of ROS detoxification is conserved in Botrytis cinerea as well. Several other phytopathogenic fungi have a corresponding K304 site and NLS present in their Srpk1, suggesting a similar mechanism of ROS detoxification and suppression of plant defense. In this article, we have presented our views on how future studies can be synthesized based on the pathway deciphered by Zhang et al. (2023, https://doi.org/10.15252/embj.2022112634).

Harnessing the Noncanonical Keap1-Nrf2 Pathway for Human Cytomegalovirus Control.

Host-derived cellular pathways can provide an unfavorable environment for virus replication. These pathways have been a subject of interest for herpesviruses, including the betaherpesvirus human cytomegalovirus (HCMV). Here, we demonstrate that a compound, ARP101, induces the noncanonical sequestosome 1 (SQSTM1)/p62-Keap1-Nrf2 pathway for HCMV suppression. ARP101 increased the levels of both LC3 II and SQSTM1/p62 and induced phosphorylation of p62 at the C-terminal domain, resulting in its increased affinity for Keap1. ARP101 treatment resulted in Nrf2 stabilization and translocation into the nucleus, binding to specific promoter sites and transcription of antioxidant enzymes under the antioxidant response element (ARE), and HCMV suppression. Knockdown of Nrf2 recovered HCMV replication following ARP101 treatment, indicating the role of the Keap1-Nrf2 axis in HCMV inhibition by ARP101. SQSTM1/p62 phosphorylation was not modulated by the mTOR kinase or casein kinase 1 or 2, indicating ARP101 engages other kinases. Together, the data uncover a novel antiviral strategy for SQSTM1/p62 through the noncanonical Keap1-Nrf2 axis. This pathway could be further exploited, including the identification of the responsible kinases, to define the biological events during HCMV replication. IMPORTANCE Antiviral treatment for human cytomegalovirus (HCMV) is limited and suffers from the selection of drug-resistant viruses. Several cellular pathways have been shown to modulate HCMV replication. The autophagy receptor sequestosome 1 (SQSTM1)/p62 has been reported to interact with several HCMV proteins, particularly with components of HCMV capsid, suggesting it plays a role in viral replication. Here, we report on a new and unexpected role for SQSTM1/p62, in HCMV suppression. Using a small-molecule probe, ARP101, we show SQSTM1/p62 phosphorylation at its C terminus domain initiates the noncanonical Keap1-Nrf2 axis, leading to transcription of genes under the antioxidant response element, resulting in HCMV inhibition in vitro. Our study highlights the dynamic nature of SQSTM1/p62 during HCMV infection and how its phosphorylation activates a new pathway that can be exploited for antiviral intervention.

Meloxicam inhibited oxidative stress and inflammatory response of LPS-stimulated bovine endometrial epithelial cells through Nrf2 and NF-κB pathways

Meloxicam is a selective cyclooxygenase-2 inhibitor and has been widely used in combination with antibiotics to alleviate uterine inflammation and provide analgesia in postpartum cows. Studies have shown that meloxicam has antioxidant and anti-inflammatory effects. However, the link between meloxicam and uterine inflammation and oxidative stress in dairy cows has not been studied. The purpose of this study was to research the effects of meloxicam (0.5 or 5 μM) on oxidative stress and inflammatory response of primary bovine endometrial epithelial cells (BEEC) stimulated by Escherichia coli lipopolysaccharide (1 μg/mL LPS). As a result, LPS stimulated the production of oxidative stress markers and the expression of inflammatory factors, accompanied by a decrease in the activity and the gene transcription of antioxidant enzymes. Co-treatment of meloxicam and LPS reduced the content of oxidative stress markers and the mRNA levels of the pro-inflammatory genes, and improved antioxidant enzyme activities and the corresponding gene expression as compared with the cells treated with LPS alone. Meloxicam attenuated the inhibitory effect of the Nrf2 pathway and the phosphorylation levels of p65 and IκBα caused by LPS. In conclusion, meloxicam alone had no effect on BEEC, but prevented oxidative stress and inflammatory response in LPS-stimulated BEEC.

Carboxymethyl chitosan regulates oxidative stress and decreases the expression levels of tumor necrosis factor α in macrophages induced by wear particles.

The aim of the present study was to determine the effects of carboxymethyl chitosan (CMC) on titanium particles-induced oxidative stress in mouse RAW264.7 macrophages. The mouse RAW264.7 macrophages were divided into four groups: (i) the control group; (ii) the CMC group received stimulation of CMC for 4h; (iii) the titanium particles group received stimulation of titanium particles for 12h; and (iv) the CMC group received pre-stimulation of CMC hydrogels for 4h followed by treatment of titanium particles for 12h. Afterwards, reactive oxygen species (ROS) level in the cells was measured by flow cytometry. A spectrophotometer was used to measure the activities of oxidases and antioxidant enzymes. Fluorescence quantitative PCR was performed to analyze mRNA levels of enzymes and tumor necrosis factor α (TNF-α). ELISA was used to detect the mass concentration of TNF-α after indicated treatment. CMC effectively suppressed titanium particles-induced oxidative stress in RAW264.7 cells, as evidenced by the decrease in intracellular ROS level, the transcription of oxidases, and TNF-α concentration as well as the increase in the transcription of antioxidant enzymes. CMC exerts a protective impact against wear particles-induced oxidative stress and reduces the release of TNF-α in RAW264.7 cells.

Identification of Nrf2/Keap1 pathway and its transcriptional regulation of antioxidant genes after exposure to microcystins in freshwater mussel Cristaria plicata

Microcystins (MC) are one of the most abundant and widely distributed cyanotoxins in aquatic systems. MC inhibits the functions of protein phosphatase 1 and 2A (PP1/2A), which can seriously affect ecosystem integrity. The NF-E2-related nuclear factor 2 (Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) signaling pathway protects against oxidative damage by activating phase II detoxification/antioxidant enzymes. Our previous study revealed that MC upregulates the expression and enhances the activities of the antioxidant enzymes by stimulating the CpNrf2 signaling pathway. In the current study, to further clarify the regulatory role of Keap1 in response to MC-induced oxidative stress in shellfish, we cloned the full-length cDNA of Keap1a and Keap1b from Cristaria plicata (designated CpKeap1a and CpKeap1b), which are 2952 and 3710 bp peptides, respectively. The amino acid sequence of CpKeap1a and CpKeap1b contained Tram-track and Bric-a-brac (BTB), Intervening region (IVR), and Double glycine repeat (DGR) domain. Additionally, CpKeap1a contained two cysteine residues analogous to Cys-273 and -288 in zebrafish, but CpKeap1b did not. Moreover, CpKeap1a and -1b formed a homodimer and heterodimer, respectively, and also formed a heterodimer with CpNrf2. In the hepatopancreas, the expression levels of CpKeap1a and -1b were the highest, but MC treatment down-regulated the expression of these proteins. Moreover, the transcription of antioxidant enzymes with antioxidant response element (ARE-driven enzymes), including CpMnSOD, CpCu/ZnSOD, CpTRX, CpPrx, CpSe-GPx, and Cpsigma-GST was upregulated by CpNrf2 in the hepatopancreas. Compared with the MC-induced group, CpKeap1a-siRNA1117 injection significantly increased the transcription of mRNAs for ARE-driven enzymes and Nrf2. CpKeap1a-siRNA1117 also enhanced the activities of antioxidation enzymes. These findings demonstrated that Keap1a negatively regulated the expression of Nrf2 protein and MC-induced oxidative stress response in C. plicata. Therefore, we speculated that CpKeap1a promoted CpNrf2 by recognizing and binding MC. These events then protected molluscs from MC-induced oxidative damage.

Ethyl Acetate Extract of Marine Algae, Halymenia durvillei, Provides Photoprotection against UV-Exposure in L929 and HaCaT Cells.

Halymenia durvillei is a red alga distributed along the coasts of Southeast Asian countries including Thailand. Previous studies have shown that an ethyl acetate fraction of H. durvillei (HDEA), containing major compounds including n-hexadecanoic acid, 2-butyl-5-hexyloctahydro-1H-indene, 3-(hydroxyacetyl) indole and indole-3-carboxylic acid, possesses high antioxidant and anti-lung cancer activities. The present study demonstrated that HDEA could protect mouse skin fibroblasts (L929) and human immortalized keratinocytes (HaCaT) against photoaging due to ultraviolet A and B (UVA and UVB) by reducing intracellular reactive oxygen species (ROS) and expressions of matrix metalloproteinases (MMP1 and MMP3), as well as increasing Nrf2 nuclear translocation, upregulations of mRNA transcripts of antioxidant enzymes, including superoxide dismutase (SOD), heme oxygenase (HMOX) and glutathione S-transferase pi1 (GSTP1), and procollagen synthesis. The results indicate that HDEA has the potential to protect skin cells from UV irradiation through the activation of the Nrf2 pathway, which leads to decreasing intracellular ROS and MMP production, along with the restoration of skin collagen.

Salicylic acid enhances the resistance of Neoporphyra haitanensis to pathogen Vibrio mediterranei 117-T6

The economically important seaweed Porphyra are often infected by various diseases during sea farming. As one of the most important phytohormones in disease resistance, the protective effect of salicylic acid (SA) on Neoporphyra haitanensis during Vibrio mediterranei 117-T6 infection was investigated by biochemical methods and transcriptomic profile analysis. The addition of exogenous SA at a moderate concentration alleviated disease caused by the pathogenic bacteria. SA enhanced the photosynthetic activity of thalli by upregulating genes of the photosynthetic system along with genes associated with carbon fixation. A moderate release of H2O2 stimulated by SA was observed, which may be due to the upregulation of the photorespiratory C2 cycle and the inhibition of catalase activity. SA treatment enhanced the activity and transcription of antioxidant enzymes in the glutathione-ascorbate (GSH-AsA) cycle involved in balancing the redox system. In addition, the levels of multiple carotenoids were increased in N. haitanensis thalli, and genes involved in their synthesis were upregulated. The results suggest that exogenous SA may enhance the defense of N. haitanensis against pathogen attack by the release of H2O2. However, to avoid oxidative stress, alga can establish redox balance by activating the GSH-AsA cycle and by regulating the generation of carotenoids. In summary, this study indicates that N. haitanensis may have evolved mechanisms to respond to SA stimulation and protect against invading pathogens. Our research can provide valuable information for the farming of healthy N. haitanensis.

Arsenic Trioxide and Venetoclax Synergize against AML Progenitors by ROS Induction and Inhibition of Nrf2 Activation.

Venetoclax (VEN) in combination with hypomethylating agents induces disease remission in patients with de novo AML, however, most patients eventually relapse. AML relapse is attributed to the persistence of drug-resistant leukemia stem cells (LSCs). LSCs need to maintain low intracellular levels of reactive oxygen species (ROS). Arsenic trioxide (ATO) induces apoptosis via upregulation of ROS-induced stress to DNA-repair mechanisms. Elevated ROS levels can trigger the Nrf2 antioxidant pathway to counteract the effects of high ROS levels. We hypothesized that ATO and VEN synergize in targeting LSCs through ROS induction by ATO and the known inhibitory effect of VEN on the Nrf2 antioxidant pathway. Using cell fractionation, immunoprecipitation, RNA-knockdown, and fluorescence assays we found that ATO activated nuclear translocation of Nrf2 and increased transcription of antioxidant enzymes, thereby attenuating the induction of ROS by ATO. VEN disrupted ATO-induced Nrf2 translocation and augmented ATO-induced ROS, thus enhancing apoptosis in LSCs. Using metabolic assays and electron microscopy, we found that the ATO+VEN combination decreased mitochondrial membrane potential, mitochondria size, fatty acid oxidation and oxidative phosphorylation, all of which enhanced apoptosis of LSCs derived from both VEN-sensitive and VEN-resistant AML primary cells. Our results indicate that ATO and VEN cooperate in inducing apoptosis of LSCs through potentiation of ROS induction, suggesting ATO+VEN is a promising regimen for treatment of VEN-sensitive and -resistant AML.

The multiple biotoxicity integrated study in grass carp (Ctenopharyngodon idella) caused by Ochratoxin A: Oxidative damage, apoptosis and immunosuppression

Ochratoxin A (OTA) is a common hazardous food contaminant that seriously endangers human and animal health. However, limited study is focused on aquatic animal. This research investigated the multiple biotoxicity of OTA on spleen (SP) and head kidney (HK) in grass carp and its related mechanism. Our data showed that, dietary supplemented with OTA above 1209 μg/kg caused histopathological damages by decreasing the number of lymphocytes and necrotizing renal parenchymal cells. Meanwhile, OTA caused oxidative damage and reduced the isoforms mRNAs transcripts of antioxidant enzymes (e.g., GPX1, GPX4, GSTO) partly due to suppressing NF-E2-related factor 2 (Nrf2). OTA triggered apoptosis through mitochondria and death receptor pathway potentially by p38 mitogen-activated protein kinase (p38MAPK) activation. Besides, OTA exacerbated inflammation by down-regulation of anti-inflammatory factor (e.g., IL-10, IL-4) and up-regulations of pro-inflammatory factors (e.g., TNF-α, IL-6), which could be ascribed to signaling meditation of Janus kinase / signal transducer and activator of transcription (JAK/STAT). Additionally, the safe upper limits of OTA were estimated to be 677.6 and 695.08 μg/kg based on the immune-related indexes (C3 contents in the SP and LZ activities in the HK, respectively). Our study has provided a wide insight for toxicological assessment of feed pollutant in aquatic animals.

Ammonia in the presence of nano titanium dioxide (nano-TiO2) induces greater oxidative damage in the gill and liver of female zebrafish

Water pollution caused by a highly hazardous chemical ammonia and a widespread application nanomaterials-nano titanium dioxide (n-TiO2) in nature water has attracted extensive concern of the world. However, the potential joint effects of the two factors are unknown. Aim to investigate the potential interactive effects of ammonia and n-TiO2 and the behind mechanisms, adult female zebrafish (Danio rerio) were co-exposed for 8 weeks by total ammonia nitrogen (TAN; 0, 3, 30 mg/L) and n-TiO2 (0, 0.1, 1 mg/L) in different combination conditions based on a full-factorial design. The analysis of absorption kinetics confirmed that n-TiO2 could absorb free ammonia (NH3) in aqueous solution and the loss rate of free NH3 increased with the rise of n-TiO2 concentration. Consistent with this, free NH3 concentrations in the gill and liver were higher in the presence of n-TiO2 compared to TAN exposure alone. The increases of MDA and PC concentrations in the gill and liver of fish indicated that TAN and n-TiO2 alone or in combination caused oxidative stress. Simultaneously, the activity and transcription of antioxidant enzymes (T-SOD, CuZn-SOD, Mn-SOD, CAT, GPx and GST) as well as antioxidant GSH contents were extensively inhibited by TAN and n-TiO2 via Nrf2-Keap1 signaling. The significant interactive effects of TAN and n-TiO2 were detected on levels of GSH, GST and gstr1 mRNA in the gill, and on levels of GSH, T-SOD, Mn-SOD, CAT levels as well as gpx1a and keap1 mRNAs in the liver, implying synergistic toxic risk of TAN and n-TiO2. The more severe histopathological alterations and higher IBR analysis in co-treatment groups further proved that the existence of n-TiO2 excavated ammonia-induced toxicity in the gill and liver, especially in liver. In conclusion, ammonia and n-TiO2 have a synergistic toxic risk of fish health because ammonia and n-TiO2 cause oxidative-antioxidative imbalance by inducing ROS overproduction.

Mycobacterium avium Subsp. hominissuis Interactions with Macrophage Killing Mechanisms.

Non-tuberculosis mycobacteria (NTM) are ubiquitously found throughout the environment. NTM can cause respiratory infections in individuals with underlying lung conditions when inhaled, or systemic infections when ingested by patients with impaired immune systems. Current therapies can be ineffective at treating NTM respiratory infections, even after a long course or with multidrug treatment regimens. NTM, such as Mycobacterium avium subspecies hominissuis (M. avium), is an opportunistic pathogen that shares environments with ubiquitous free-living amoeba and other environmental hosts, possibly their evolutionary hosts. It is highly likely that interactions between M. avium and free-living amoeba have provided selective pressure on the bacteria to acquire survival mechanisms, which are also used against predation by macrophages. In macrophages, M. avium resides inside phagosomes and has been shown to exit it to infect other cells. M. avium’s adaptation to the hostile intra-phagosomal environment is due to many virulence mechanisms. M. avium is able to switch the phenotype of the macrophage to be anti-inflammatory (M2). Here, we have focused on and discussed the bacterial defense mechanisms associated with the intra-phagosome phase of infection. M. avium possesses a plethora of antioxidant enzymes, including the superoxide dismutases, catalase and alkyl hydroperoxide reductase. When these defenses fail or are overtaken by robust oxidative burst, many other enzymes exist to repair damage incurred on M. avium proteins, including thioredoxin/thioredoxin reductase. Finally, M. avium has several oxidant sensors that induce transcription of antioxidant enzymes, oxidation repair enzymes and biofilm- promoting genes. These expressions induce physiological changes that allow M. avium to survive in the face of leukocyte-generated oxidative stress. We will discuss the strategies used by M. avium to infect human macrophages that evolved during its evolution from free-living amoeba. The more insight we gain about M. avium’s mode of pathogenicity, the more targets we can have to direct new anti-virulence therapies toward.

Structural Characterization of KEAP1 R415K to Guide the Design of KEAP1‐Nrf2 Peptide Inhibitors

Oxidative stress can cause extensive damage to DNA, proteins, and lipids. Thus, long-term oxidative stress has been linked to many diseases including cancer and Alzheimer's disease. The highly-charged binding interface between regulatory protein Kelch-like ECH-associated protein 1 (KEAP1) and transcription factor nuclear erythroid factor 2-like 2 (Nrf2) is a common drug target for diseases related to oxidative stress. In the absence of oxidative stress, KEAP1 binds with high affinity to the DxETGE motif of Nrf2. Oxidative stress causes KEAP1 to release Nrf2, allowing it to translocate to the nucleus and upregulate the transcription of antioxidant enzymes. Our current peptide inhibitors of this protein-protein interaction (PPI) are modeled after the DxETGE motif and have exhibited low cell permeability, prompting the bioisosteric replacement of charged residues. We previously identified Arg415 as the KEAP1 residue with the most significant contribution to binding energy at the KEAP1-Nrf2 interface. It has been observed that the R415A mutation abolishes Nrf2 binding, and so a more conservative KEAP1 R415K variant was characterized to better understand the interactions between this residue and residue Glu79 on Nrf2. The variant was characterized using X-ray crystallography, differential scanning fluorimetry, and the binding was assessed using a competitive fluorescence anisotropy binding assay. This mutation caused a 40-fold decrease in binding affinity, which suggested that retaining the charge alone was not sufficient to restore wild-type binding affinity. KEAP1 R415K was successfully crystallized in 8% w/v polyethylene glycol 20,000, 0.2 M imidazole, and 0.01 M nickel (II) chloride hexahydrate, and the crystals diffracted to a resolution of 2.43 Å. Structures of the unliganded R415K Kelch domain were determined using molecular replacement. Optimization of the crystals toward the structure determination of the R415K variant bound to peptide inhibitors is underway. These liganded structures can then be used to assess the binding interactions between KEAP1 Arg415 and Nrf2 Glu79 and guide future peptide inhibitor optimizations to improve their potency and bioavailability.

Dose-Dependent Physiological and Transcriptomic Responses of Lettuce (Lactuca sativa L.) to Copper Oxide Nanoparticles-Insights into the Phytotoxicity Mechanisms.

Understanding the complex mechanisms involved in plant response to nanoparticles (NPs) is indispensable in assessing the environmental impact of nano-pollutants. Plant leaves can directly intercept or absorb NPs deposited on their surface; however, the toxicity mechanisms of NPs to plant leaves are unclear. In this study, lettuce leaves were exposed to copper oxide nanoparticles (CuO-NPs, 0, 100, and 1000 mg/L) for 15 days, then physiological tests and transcriptomic analyses were conducted to evaluate the negative impacts of CuO-NPs. Both physiological and transcriptomic results demonstrated that CuO-NPs adversely affected plant growth, photosynthesis, and enhanced reactive oxygen species (ROS) accumulation and antioxidant system activity. The comparative transcriptome analysis showed that 2270 and 4264 genes were differentially expressed upon exposure to 100 and 1000 mg/L CuO-NPs. Gene expression analysis suggested the ATP-binding cassette (ABC) transporter family, heavy metal-associated isoprenylated plant proteins (HIPPs), endocytosis, and other metal ion binding proteins or channels play significant roles in CuO-NP accumulation by plant leaves. Furthermore, the variation in antioxidant enzyme transcript levels (POD1, MDAR4, APX2, FSDs), flavonoid content, cell wall structure and components, and hormone (auxin) could be essential in regulating CuO-NPs-induced stress. These findings could help understand the toxicity mechanisms of metal NPs on crops, especially NPs resulting from foliar exposure.

The CD36 Ligand-Promoted Autophagy Protects Retinal Pigment Epithelial Cells from Oxidative Stress.

The retinal pigment epithelium (RPE) performs many functions that maintain photoreceptor health. Oxidative damage to the RPE is a critical component in the pathogenesis of eye diseases such as age-related macular degeneration (AMD). Ligands of the cluster of differentiation 36 (CD36) have previously preserved photoreceptor integrity in mouse models of AMD. The cytoprotective effect of the CD36 ligand MPE-001 on RPE cells has now been elucidated employing a model of oxidative stress. Sodium iodate (NaIO3) induced formation of reactive oxygen species and apoptosis in human RPE cells, which were decreased by MPE-001 without affecting antioxidant enzyme transcription. Immunoblotting and immunostaining assays showed a restorative effect of MPE-001 on the autophagic flux disrupted by NaIO3, which was associated with an increase in syntaxin 17-positive mature autophagosomes. The cytoprotective effect of MPE-001 was completely abolished by the autophagy inhibitors wortmannin and bafilomycin A1. In conclusion, we report for the first time an autophagy-dependent protection of RPE cells from oxidative stress by a CD36 ligand.

Effect of manganese supplementation on the carcass traits, meat quality, intramuscular fat, and tissue manganese accumulation of Pekin duck

Manganese (Mn) is a trace element present in all tissues and is essential for animal growth and health; it also has an antioxidant capacity in tissues. The effect of Mn on meat quality and the mechanism of fat deposition of the breast muscle is still unclear. Therefore, the present study aimed to investigate the effect of Mn supplementation on the growth performance, meat quality, the activity and transcription of antioxidant enzymes, and fatty acid profile in the breast muscle, and the Mn deposition in tissues of Pekin ducks. A total of 896 one-day-old Pekin ducks were allocated into 7 groups, with 8 replicates, each replicate containing 16 ducks. The treatment diets consisted of basal diet supplemented with manganese sulfate at levels 30, 60, 90, 120, 150, 240 mg/kg (as Mn). Results showed that ducks fed diets supplemented with Mn had no effect on the growth performance but decrease in the feed-to-gain ratio of day 1–14 (P < 0.01). Dietary Mn increased significantly the a∗ (redness) value of the duck breast meat at 24 h and intramuscular fat (P < 0.05), and decreased drip loss and shear force of the breast meat (P < 0.05). Manganese supplement significantly reduced the malondialdehyde content (P < 0.05), and significantly increased the mRNA expressions of manganese superoxide dismutase, thioredoxin 2, peroxiredoxin 3, and catalase (P < 0.05). About the fatty acid profile, dietary Mn increased (P < 0.05) the proportions of the C20 family. Manganese accumulation in the heart, breast muscle, and tibia was increased with Mn supplementation (P < 0.05), and Mn content of the heart conforms to the quadratic curve. Besides, Mn supplementation notably increased mRNA expression in genes involved in lipogenesis and deposition and decreased in genes associated with lipolytic in the breast muscle. These findings reveal that dietary Mn could improve meat quality and enhance antioxidant activity and intramuscular fat, which via regulated gene expression involved in lipogenesis and lipolytic.

A Melanin-Related Phenolic Polymer with Potent Photoprotective and Antioxidant Activities for Dermo-Cosmetic Applications.

Eumelanins, the dark variant of skin pigments, are endowed with a remarkable antioxidant activity and well-recognized photoprotective properties that have been ascribed to pigment components derived from the biosynthetic precursor 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Herein, we report the protective effect of a polymer obtained starting from the methyl ester of DHICA (MeDHICA-melanin) against Ultraviolet A (UVA)-induced oxidative stress in immortalized human keratinocytes (HaCaT). MeDHICA-melanin was prepared by aerial oxidation of MeDHICA. At concentrations as low as 10 µg/mL, MeDHICA-melanin prevented reactive oxygen species accumulation and partially reduced glutathione oxidation in UVA-irradiated keratinocytes. Western blot experiments revealed that the polymer is able to induce the translocation of nuclear factor erythroid 2–related factor 2 (Nrf-2) to the nucleus with the activation of the transcription of antioxidant enzymes, such as heme-oxygenase 1. Spectrophotometric and HPLC analysis of cell lysate allowed to conclude that a significant fraction (ca. 7%), consisting mainly of the 4,4′-dimer of MeDHICA (ca. 2 μM), was internalized in the cells. Overall these data point to the potential use of MeDHICA-melanin as an antioxidant for the treatment of skin damage, photoaging and skin cancers.

Melatonin alleviates iron stress by improving iron homeostasis, antioxidant defense and secondary metabolism in cucumber

Iron is an essential element for plants and animals; however, deficit or excess iron can result in stress on plants. In this study, we unveiled the mechanisms of shoot-based tolerance to iron toxicity by melatonin (N-acetyl-5-methoxytryptamine) in cucumber plants. Both low and high iron (Fe) supply in hydroponics decreased growth and biomass accumulation, induced chlorosis and oxidative stress, and reduced chlorophyll content, photosynthesis rate and transpiration rate in cucumber leaves. Notably, the negative effect of low-Fe was more profound than that of high-Fe treatment. However, exogenous melatonin application alleviated those inhibitions in growth, biochemical and physiological parameters, which entailed an elevation of endogenous melatonin content and a reduction of electrolyte leakage, reactive oxygen species accumulation and lipid peroxidation by improving the activity and transcripts of antioxidant enzymes and secondary metabolism-related enzymes, and concentrations of phenols and flavonoids under low and high iron conditions after melatonin treatment. Analysis of iron content in leaves and roots revealed that melatonin significantly increased the iron content under low-Fe conditions, but it decreased the same under high-Fe conditions. Moreover, melatonin increased the transcript levels of FRO2 and IRT1 under low-Fe, but it decreased those transcripts under high-Fe, suggesting that melatonin plays a dual role in iron uptake under low and high iron conditions. This study expands the stress ameliorative role of melatonin in plants and may have potential implications in agronomic management of crops in low and high iron prone soils.