Peroxiredoxin Research Articles

Protective Effects of Peroxiredoxin 6 in Pro-Inflammatory Response Model Using Raw 264.7 Macrophages.

The aim of the work was to study effects of peroxiredoxin 6 (PRDX6), a recombinant antioxidant protein, on the level of pro-inflammatory responses of RAW 264.7 macrophages to endotoxin exposure. Addition of LPS to the RAW 264.7 cell culture medium expectedly increased production of TNF-α, and addition of PRDX6 led to a significant (15-20%) decrease in its production. The level of production of another pro-inflammatory cytokine, IL-1β, which was significantly activated by endotoxin, was completely normalized under the PRDX6 action. Moreover, addition of PRDX6 reduced production of reactive oxygen species (ROS) induced by endotoxin and also prevented overexpression of the iNos gene in the RAW 264.7 cells. The results showed that PRDX6 had a suppressive effect on the expression of Nrf-2 gene and production of the transcription factor NRF-2 during the first 6 h of cell cultivation. Addition of endotoxin caused activation of the NF-κB and SAPK/JNK signaling cascades, while in the presence of PRDX6, activity of these signaling cascades decreases. It is known that the pro-inflammatory response of cells caused by exposure to bacterial LPS leads to activation of apoptosis and elimination of the damaged cells. Our studies confirm this, since exposure to LPS led to activation of the expression of P53 gene, a marker of apoptosis. Peroxiredoxin 6 added within the first hours of the development of acute pro-inflammatory response suppressed the P53 gene expression, indicating protective effect of PRDX6 that reduced apoptosis in the RAW 264.7 macrophages.

Conoidin A, a Covalent Inhibitor of Peroxiredoxin 2, Reduces Growth of Glioblastoma Cells by Triggering ROS Production.

Compounds that cause oxidative stress have recently gained considerable interest as potential anticancer treatment modalities. Nevertheless, their efficiency may be diminished by the antioxidant systems often upregulated in cancer cells. Peroxiredoxins (PRDXs) are antioxidant enzymes that scavenge peroxides and contribute to redox homeostasis. They play a role in carcinogenesis and are upregulated in several cancer types. Here, we assessed the expression pattern of PRDX1 and PRDX2 in glioblastoma (GBM) and examined the efficacy of their inhibitors in GBM cell lines and patient-derived GBM cells. Both PRDX1 and PRDX2 were upregulated in GBM compared to non-tumor brain tissues and their considerable amounts were observed in GBM cells. Adenanthin, a compound inhibiting PRDX1 activity, slightly decreased GBM cell viability, while conoidin A (CONA), a covalent PRDX2 inhibitor, displayed high toxicity in GBM cells. CONA elevated the intracellular reactive oxygen species (ROS) level. Pre-treatment with an ROS scavenger protected cells from CONA-induced death, indicating that ROS accumulation plays a crucial role in this phenomenon. Menadione or celecoxib, both of which are ROS-inducing agents, potentiated the anticancer activity of CONA. Collectively, our results unveil PRDX1 and PRDX2 as potential targets for GBM therapy, and substantiate the further exploration of their inhibitors.

Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.

Damage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells. Cells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining. Our results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression. Our findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.

The PRX-1/TLR4 axis promotes hypoxia-induced radiotherapy resistance in non-small cell lung cancer by targeting the NF-κB/p65 pathway

Hypoxic lung cancer cells are highly resistant to radiation. Peroxiredoxin-1 (PRX-1), a transcriptional coactivator that enhances the DNA-binding activity of serum reactive factor, has been identified as a target for radiotherapy sensitization, but the underlying molecular mechanism remains unclear. This study aimed to investigate the influence of PRX-1 on radiotherapy sensitivity in hypoxic tumors. Hypoxic lung cancer cells exhibited radiotherapy-resistant phenotypes after irradiation, including increased proliferation, DNA damage repair, cell migration, invasion and stemness. Radio-resistant hypoxic lung cancer cells showed high expression levels of PRX-1. Furthermore, we observed that PRX-1 bound to the promoter region of TRL4 (−300 to −600) and promoted its transcription and expression and that PRX-1/TRL4 activated the NF-κB/p65 signaling pathway. Increased radiotherapy resistance of hypoxic lung cancer cells increased their ability to proliferate, migrate, and maintain stemness in vivo and in vitro. These findings suggest that PRX-1/TRL4 could be used as a target for the treatment of radiotherapy-resistant lung cancer cells and further provide a theoretical basis for the clinical treatment of hypoxic lung cancer cells.

Cytotoxicity of alkaline serine protease (ASPNJ) on Jurkat cells and its correlation with changes in the expression of membrane-associated proteins.

We aim to study the inhibitory effect of alkaline serine protease (ASPNJ) on lymphocytic leukemia Jurkat cells and its related mechanism through examining the expression of membrane proteins or membrane-associated proteins. MTT assay and trypan blue staining were used to detect the inhibitory effect of ASPNJ on the proliferation and growth of Jurkat cells. Wright-Giemsa staining was used to observe the effect of ASPNJ on the morphology of Jurkat cells. The effect of ASPNJ on Jurkat cell apoptosis was detected by flow cytometry. Two-dimensional electrophoresis-mass spectrometry (2-DE-MS) was used to detect and identify the differentially expressed proteins of Jurkat cells treated with ASPNJ (4 μg/mL, 3 h), of which three were selected and verified by Western blot. ASPNJ significantly inhibited the proliferation of leukemia cells (Raji, U937, and Jurkat), caused obvious morphological changes, and induced apoptosis of Jurkat cells. ASPNJ also increased the sensitivity of Jurkat cells to vincristine (VCR). Seven differentially expressed proteins were obtained through 2DE-MS, of which Peroxiredoxin-6 (PRDX6), Calcium-binding protein (CHP1), and 40S ribosomal protein SA (RPSA) were validated. ASPNJ can cause significant toxic effects on Jurkat cells and enhance the effects of VCR. The mechanism of action of ASPNJ on Jurkat cells may be related to differentially expressed proteins such as PRDX6. This study provides a new experimental basis and direction for antileukemia research.

Ferrostatin-1 attenuates brain injury in animal model of subarachnoid hemorrhage via phospholipase A2 activity of PRDX6.

Subarachnoid hemorrhage (SAH) is an acute catastrophic neurological disorder with high morbidity and mortality. Ferroptosis is one of the pathophysiological processes during secondary brain injury of SAH, which could be inhibited by ferrostatin-1 (Fer-1) effectively. Peroxiredoxin6 (PRDX6) is an antioxidant protein and is currently proven to be associated with lipid peroxidation in ferroptosis except in GSH/GPX4 and FSP1/CoQ10 antioxidant systems. However, the alteration and function of PRDX6 in SAH are still unknown. In addition, whether PRDX6 is involved in the neuroprotection of Fer-1 in SAH is yet to be investigated. Endovascular perforation was employed to induce the SAH model. Fer-1 and in vivo siRNA aiming to knockdown PRDX6 were administrated intracerebroventricularly to investigate relevant regulation and mechanism. We confirmed the inhibition of ferroptosis and neuroprotection from brain injury by Fer-1 in SAH. The induction of SAH reduced the expression of PRDX6, which could be alleviated by Fer-1. Accordingly, dysregulated lipid peroxidation indicated by GSH and MDA was improved by Fer-1, which was counteracted by si-PRDX6. Similarly, the neuroprotection of Fer-1 in SAH was diminished by the knockdown of PRDX6 and the administration of a calcium-independent phospholipase A2 (iPLA2) inhibitor. PRDX6 is involved in ferroptosis induced by SAH and is associated with Fer-1 neuroprotection from brain injury via its iPLA2 activity.

Peroxinredoxin 6 reduction accelerates cigarette smoke extract‑induced senescence by regulating autophagy in BEAS‑2B cells.

Cigarette smoke (CS)-induced accelerated senescence and insufficient autophagy has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Peroxiredoxin (PRDX) 6 is a protein with prevalent antioxidant capacity. Previous studies indicate that PRDX6 could activate autophagy and alleviate senescence in other diseases. The present study investigated whether PRDX6-regulated autophagy was involved in the regulation of CS extract (CSE)-induced BEAS-2B cell senescence via the knockdown of PRDX6 expression. Furthermore, the present study evaluated the mRNA levels of PRDX6, autophagy and senescence-associated genes in the small airway epithelium from patients with COPD by analyzing the GSE20257 dataset from the Gene Expression Omnibus database. The results demonstrated that CSE reduced PRDX6 expression levels and transiently induced the activation of autophagy, followed by the accelerated senescence of BEAS-2B cells. Knockdown of PRDX6 induced autophagy degradation and accelerated senescence in CSE-treated BEAS-2B cells. Furthermore, autophagy inhibition by 3-Methyladenine increased P16 and P21 expression levels, while autophagy activation by rapamycin reduced P16 and P21 expression levels in CSE-treated BEAS-2B cells. The GSE20257 dataset revealed that patients with COPD had lower PRDX6, sirtuin (SIRT) 1 and SIRT6 mRNA levels, and higher P62 and P16 mRNA levels compared with non-smokers. P62 mRNA was significantly correlated with P16, P21 and SIRT1, which indicated that insufficient autophagic clearance of damaged proteins could be involved in accelerated cell senescence in COPD. In conclusion, the present study demonstrated a novel protective role for PRDX6 in COPD. Furthermore, a reduction in PRDX6 could accelerate senescence by inducing autophagy impairment in CSE-treated BEAS-2B cells.

Panax notoginseng alleviates oxidative stress through miRNA regulations based on systems biology approach

BackgroundHerbal medicine Sanqi (SQ), the dried root or stem of Panax notoginseng (PNS), has been reported to have anti-diabetic and anti-obesity effects and is usually administered as a decoction for Chinese medicine. Alternative to utilizing PNS pure compound for treatment, we are motivated to propose an unconventional scheme to investigate the functions of PNS mixture. However, studies providing a detailed overview of the transcriptomics-based signaling network in response to PNS are seldom available.MethodsTo explore the reasoning of PNS in treating metabolic disorders such as insulin resistance, we implemented a systems biology-based approach with RNA sequencing (RNA-seq) and miRNA sequencing data to elucidate key pathways, genes and miRNAs involved.ResultsFunctional enrichment analysis revealed PNS up-regulating oxidative stress-related pathways and down-regulating insulin and fatty acid metabolism. Superoxide dismutase 1 (SOD1), peroxiredoxin 1 (PRDX1), heme oxygenase-1 (Hmox1) and glutamate cysteine ligase (GCLc) mRNA and protein levels, as well as related miRNA levels, were measured in PNS treated rat pancreatic β cells (INS-1). PNS treatment up-regulated Hmox1, SOD1 and GCLc expression while down-regulating miR-24-3p and miR-139-5p to suppress oxidative stress. Furthermore, we verified the novel interactions between miR-139-5p and miR-24-3p with GCLc and SOD1.ConclusionThis work has demonstrated the mechanism of how PNS regulates cellular molecules in metabolic disorders. Therefore, combining omics data with a systems biology strategy could be a practical means to explore the potential function and molecular mechanisms of Chinese herbal medicine in the treatment of metabolic disorders.

Effect of PRDX6 gene polymorphism on susceptibility to chronic obstructive pulmonary disease in the Chinese Han population.

To explore the relationship of peroxiredoxin6 (PRDX6) tag-single nucleotide polymorphisms (SNPs) with susceptibility to chronic obstructive pulmonary disease (COPD) in the Chinese Han population. A total of 502 patients with COPD and 481 healthy controls from nine hospitals in China were enrolled in this study. The PRDX6 tag-SNPs were identified by linkage disequilibrium (LD) analysis in 30 healthy controls. The associations between identified tag-SNPs and COPD risk were further evaluated. Four PRDX6 tag-SNPs, including rs7314, rs34619706, rs33951697, and rs4382766, were identified in 30 healthy controls. Moreover, in the allele model, there was no statistical difference in locus in PRDX6 between patients with COPD and healthy controls (P > 0.05). However, in the recessive model, rs33951697 locus in PRDX6 gene carrier with T/T had an increased risk of COPD (odds ratio [OR] = 2.59, 95% CI = 1.06-6.33, P = 0.028). Furthermore, in the relevance analysis between genetic polymorphisms and smoking behavior and lung function indexes, we found that the number of smoked cigarettes per day and FEV1/FVC differed among different genotypes of PRDX6, rs4382766, and rs7314 (P < 0.05). PRDX6 gene polymorphism with smoking status may contribute to the etiology of COPD in the Chinese Han population.

Polygenic Variants Linked to Oxidative Stress and the Antioxidant System Are Associated with Type 2 Diabetes Risk and Interact with Lifestyle Factors.

Oxidative stress is associated with insulin resistance and secretion, and antioxidant systems are essential for preventing and managing type 2 diabetes (T2DM). This study aimed to explore the polygenic variants linked to oxidative stress and the antioxidant system among those associated with T2DM and the interaction of their polygenic risk scores (PRSs) with lifestyle factors in a large hospital-based cohort (n = 58,701). Genotyping, anthropometric, biochemical, and dietary assessments were conducted for all participants with an average body mass index of 23.9 kg/m2. Genetic variants associated with T2DM were searched through genome-wide association studies in participants with T2DM (n = 5383) and without T2DM (n = 53,318). The Gene Ontology database was searched for the antioxidant systems and oxidative stress-related genes among the genetic variants associated with T2DM risk, and the PRS was generated by summing the risk alleles of selected ones. Gene expression according to the genetic variant alleles was determined on the FUMA website. Food components with low binding energy to the GSTA5 protein generated from the wildtype and mutated GSTA5_rs7739421 (missense mutation) genes were selected using in silico analysis. Glutathione metabolism-related genes, including glutathione peroxidase (GPX)1 and GPX3, glutathione disulfide reductase (GSR), peroxiredoxin-6 (PRDX6), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase alpha-5 (GSTA5), and gamma-glutamyltransferase-1 (GGT1), were predominantly selected with a relevance score of >7. The PRS related to the antioxidant system was positively associated with T2DM (ORs = 1.423, 95% CI = 1.22-1.66). The active site of the GASTA proteins having valine or leucine at 55 due to the missense mutation (rs7739421) had a low binding energy (<-10 kcal/mol) similarly or differently to some flavonoids and anthocyanins. The PRS interacted with the intake of bioactive components (specifically dietary antioxidants, vitamin C, vitamin D, and coffee) and smoking status (p < 0.05). In conclusion, individuals with a higher PRS related to the antioxidant system may have an increased risk of T2DM, and there is a potential indication that exogenous antioxidant intake may alleviate this risk, providing insights for personalized strategies in T2DM prevention.

#5219 PEROXIREDOXINS ARE RELATED TO EGFR DECLINE IN IGA NEPHROPATHY, MEMBRANOUS NEPHROPATHY AND LUPUS NEPHRITIS: A 5-YEAR FOLLOW-UP

Abstract Background and Aims Oxidative stress (OS) is known as a disturbance between pro- and antioxidant factors, that may lead to DNA or protein oxidation, resulting in cellular damage. Peroxiredoxins (PRDXs) are enzymes with an antioxidant properties. They play dominant role in regulation of cellular peroxide levels, which is crucial in maintaining organisms’ oxidative balance. PRDXs types 1-5 are involved in the pathophysiology of various diseases (e.g. cancer, inflammatory, or renal). We aim to study their role as a prognostic marker of eGFR changes in IgA nephropathy (IgAN), lupus nephritis (LN) and membranous nephropathy (MN) patients. Method This is a retrospective 5-year-follow-up study of 108 IgAN, MN and LN patients, in whom PRDX 1-5 serum levels were assessed with ELISA in 2017. In 2022, we were able to collect the data out of 80 patients: IgAN (n=36); MN (n=22) and LN (n=22). The remaining 28 were considered as lost-to-follow-up, namely: lack of any medical results within last 2 years (n=23); on dialysis (n=2); after renal transplantation (n=2); deceased (n=1). We classified patients depending on the changes of eGFR (decline/stable/increase; Fig. 1) between baseline and after 5 year follow-up and the PRDX levels (high/medium/low; Table 1). The Mann Whitney-U, Kruskal-Wallis and Chi-Square Test were used for statistical analysis, the p-values &amp;lt;0.05 were considered significant. Results Baseline PRDX measurements indicated, that the mean concentrations of serum PRDX 1-5 differ between the glomerulonephropathies (GN). In 2022, we observed significantly different (P = .012) change of eGFR depending on the GN type. 16/80 individuals had eGFR decline and 75% of them were diagnosed with IgAN. The MN and LN groups had increase of eGFR in the last 5 years in most cases, probably as a result of the treatment. We verified if follow-up eGFR was associated with baseline PRDX levels. The 2022 eGFR differed significantly between the GNs depending on the level of serum PRDX 2, 4 and 5 at baseline (P = .002; P = .009; P = .006, respectively, Table 1). Conclusion Our results showed the link between serum PRDXs concentration and IgAN, MN and LN follow-up. Importantly, selected PRDXs’ might be used to predict eGFR decline, particularly in IgAN and LN.

Knockdown of circSOD2 ameliorates osteoarthritis progression via the miR-224-5p/PRDX3 axis

BackgroundAlthough the implications of circular RNAs (circRNAs) with the progression of diverse pathological conditions have been reported, the circRNA players in osteoarthritis (OA) are barely studied.MethodsIn this study, twenty-five OA patients who received arthroplasty were recruited for cartilage tissue collection. Public circRNA microarray data from Gene Expression Omnibus was retrieved for circRNA identification. An in vitro cell model of OA-related damages was constructed by treating human chondrocytes (CHON-001 cell line) with IL-1β, and circSOD2 siRNA was used to silence circSOD2 expression to study its functional role in apoptosis, inflammatory responses, and extracellular matrix (ECM) degradation. Besides, we investigated the functional interactions among circSOD2, miR-224-5p, and peroxiredoxin 3 (PRDX3) by luciferase reporter assay, RNA-immunoprecipitation assay, and quantitative reverse transcription polymerase chain reaction.ResultsOur findings revealed the overexpression of circSOD2 in the OA cartilage and cell samples, and circSOD2 knockdown alleviated ECM degradation, inflammation, and apoptosis in CHON-001 cell model. In addition, our findings suggested the regulatory function of circSOD2 knockdown on miR-224-5p expression, while miR-224-5p was capable of downregulating PRDX3 expression. The co-transfection of miR-224-5p inhibitor or pcDNA-PRDX3 could prevent the effect of circSOD2 knockdown.ConclusionHence, our results demonstrated that knockdown of circSOD2 may serve as an intervention strategy to alleviate OA progression through modulating miR-224-5p/PRDX3 signaling axis.

PRDX1 negatively regulates bleomycin-induced pulmonary fibrosis via inhibiting the epithelial-mesenchymal transition and lung fibroblast proliferation in vitro and in vivo

BackgroundPulmonary fibrosis is a major category of end-stage changes in lung diseases, characterized by lung epithelial cell damage, proliferation of fibroblasts, and accumulation of extracellular matrix. Peroxiredoxin 1 (PRDX1), a member of the peroxiredoxin protein family, participates in the regulation of the levels of reactive oxygen species in cells and various other physiological activities, as well as the occurrence and development of diseases by functioning as a chaperonin.MethodsExperimental methods including MTT assay, morphological observation of fibrosis, wound healing assay, fluorescence microscopy, flow cytometry, ELISA, western blot, transcriptome sequencing, and histopathological analysis were used in this study.ResultsPRDX1 knockdown increased ROS levels in lung epithelial cells and promoted epithelial-mesenchymal transition (EMT) through the PI3K/Akt and JNK/Smad signalling pathways. PRDX1 knockout significantly increased TGF-β secretion, ROS production, and cell migration in primary lung fibroblasts. PRDX1 deficiency also increased cell proliferation, cell cycle circulation, and fibrosis progression through the PI3K/Akt and JNK/Smad signalling pathways. BLM treatment induced more severe pulmonary fibrosis in PRDX1-knockout mice, mainly through the PI3K/Akt and JNK/Smad signalling pathways.ConclusionsOur findings strongly suggest that PRDX1 is a key molecule in BLM-induced lung fibrosis progression and acts through modulating EMT and lung fibroblast proliferation; therefore, it may be a therapeutic target for the treatment of BLM-induced lung fibrosis.

Alterations in the expression pattern of RBC membrane associated proteins (RMAPs) in whole body γ-irradiated Sprague Dawley rats

Purpose Peripheral blood serum/plasma proteins are frequently studied for their potential use as radiation exposure biomarkers. Here we report RBC membrane associated proteins (RMAPs), which show alterations in expression level following whole-body γ-irradiation of rats at sub-lethal/lethal doses. Materials and Methods RBCs from peripheral blood of Sprague Dawley rats were segregated using the Ficoll-Hypaque method, and membrane fractions were hypotonically isolated at various time points (6 h, 24 h, 48 h) after γ-irradiation at 2 Gy, 5 Gy, and 7.5 Gy doses. Following purification of proteins from these fractions, two-dimensional electrophoresis (2-DE) was carried out. Treatment induced differentially expressed protein spots (≥2 fold increase/decrease) were picked up, trypsinized, and identified using LC-MS/MS analysis. Western immunoblots using protein specific antibodies were used to confirm the results. Gene ontology and interactions of these proteins were also studied. Results From a number of differentially expressed radiation-responsive 2-DE protein spots detected, eight were identified unequivocally using LC-MS/MS. Out of these, actin, cytoplasmic 1 (ACTB) showed detectable yet insignificant variation (<50%) in expression. In contrast, peroxiredoxin-2 (PRDX2) and 26S proteasome regulatory subunit RPN11 (PSMD14) were the two most prominently over-expressed proteins. Five more proteins, namely tropomyosin alpha-3 chain (TPM3), exosome component 6 (EXOSC6), isoform 4 of tropomyosin alpha-1 chain (TPM1), serum albumin (ALB), and the 55 kDa erythrocyte membrane protein (P55) showed distinct alteration in their expression at different time-points and doses. ALB, EXOSC6, and PSMD14 were the most responsive at 2 Gy, albeit at different time-points. While EXOSC6 and PSMD14 showed maximum over-expression (5-12 fold) at 6 h post-irradiation, ALB expression increased progressively (4 up to 7 fold) from 6 h to 48 h. TPM1 showed over-expression (2-3 fold) at all doses and time-points tested. TPM3 showed a dose-dependent response at all time-points studied; with no variation at 2 Gy, ∼2 fold increase at 5 Gy, and 3-6 fold at the highest dose used (7.5 Gy). The p55 protein was over-expressed (∼2.5 fold) only transiently at 24 h following the lethal (7.5 Gy) dose. Conclusion This is the first study to report γ-radiation induced alterations in the RBC membrane associated proteins. We are further evaluating the potential of these proteins as radiation biomarkers. Due to the abundance and easy use of RBCs, this approach can prove very useful for detecting ionizing radiation exposure.

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines.

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3β inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the "cadherin switch". These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies.

Ficus deltoidea Leaf Alters Oxidative Stress, Protein Homeostasis and Ubiquitin-Proteasome Pathways in Fatty Acid-Induced Cell Line.

Ficus deltoidea (mistletoe fig) is a shrub well known among locals in Malaysia primarily for its treatment of toothaches, colds and wounds. The aim of this study is to determine the potential of leaves, sourced from three different varieties of F. deltoidea, to exhibit antioxidant activity, a reduction of lipid concentration, and protein expression in steatosis-induced liver cell lines. The leaves of three F. deltoidea varieties, namely Ficus deltoidea var. angustifolia, Ficus deltoidea var. trengganuensis and Ficus deltoidea var. kunstleri, were subjected to water extraction. The resulting crude extracts were fractionated using water and ethyl acetate. Palmitic acid was used to induce lipid accumulation (steatosis) in human liver (WRL68) cells, before all the samples were tested for their lipid-reducing activity. Several proteomic approaches were incorporated. The changes in protein expression were determined using 2-dimensional gel electrophoresis separation, whereas identification of our protein spots of interest was carried out via matrix-assisted laser desorption/ionization time-of-flight. Ficus deltoidea var. kunstleri alone demonstrated the ability to reduce lipids at the highest tested concentration (200 µg/mL) and was, therefore, used for subsequent experiments. Treatment with Ficus deltoidea var. kunstleri was found to restore redox status by increasing superoxide dismutase and glutathione peroxidase amounts and decreasing malondialdehyde formation. Six proteins were successfully identified; these were heat shock protein beta-1 (HSPB1), proteasome subunit alpha type 1 (PSMA1), glutathione S-transferase omega 1 (GSTO1), peroxiredoxin-1 (PRDX1), histone H2B (HIST1H2BD) and ubiquitin c-terminal hydrolase L3 (UCHL3). Through bioinformatics analysis, it was found that these proteins were significantly involved in specific pathways such as oxidative stress (PRDX1 and GSTO1), protein homeostasis (HSPB1) and degradation (UCHL3 and PSMA1). F. deltoidea pretreatment was shown to reduce lipid accumulation, thus improving the redox status and protein homeostasis. This suggests the role of F. deltoidea as a preventive mechanism in non-alcohol fatty liver disease.

The contribution of glutathione peroxidases to chloroplast redox homeostasis in Arabidopsis

Oxidizing signals mediated by the thiol-dependent peroxidase activity of 2-Cys peroxiredoxins (PRXs) plays an essential role in fine-tuning chloroplast redox balance in response to changes in light intensity, a function that depends on NADPH-dependent thioredoxin reductase C (NTRC). In addition, plant chloroplasts are equipped with glutathione peroxidases (GPXs), thiol-dependent peroxidases that rely on thioredoxins (TRXs). Despite having a similar reaction mechanism than 2-Cys PRXs, the contribution of oxidizing signals mediated by GPXs to the chloroplast redox homeostasis remains poorly known. To address this issue, we have generated the Arabidopsis (Arabidopsis thaliana) double mutant gpx1gpx7, which is devoid of the two GPXs, 1 and 7, localized in the chloroplast. Furthermore, to analyze the functional relationship of chloroplast GPXs with the NTRC-2-Cys PRXs redox system, the 2cpab-gpx1gpx7 and ntrc-gpx1gpx7 mutants were generated. The gpx1gpx7 mutant displayed wild type-like phenotype indicating that chloroplast GPXs are dispensable for plant growth at least under standard conditions. However, the 2cpab-gpx1gpx7 showed more retarded growth than the 2cpab mutant. The simultaneous lack of 2-Cys PRXs and GPXs affected PSII performance and caused higher delay of enzyme oxidation in the dark. In contrast, the ntrc-gpx1gpx7 mutant combining the lack of NTRC and chloroplast GPXs behaved like the ntrc mutant indicating that the contribution of GPXs to chloroplast redox homeostasis is independent of NTRC. Further supporting this notion, in vitro assays showed that GPXs are not reduced by NTRC but by TRX y2. Based on these results, we propose a role for GPXs in the chloroplast redox hierarchy.

CircZNF215 promotes tumor growth and metastasis through inactivation of the PTEN/AKT pathway in intrahepatic cholangiocarcinoma

BackgroundIncreasing evidence shows that circular RNAs (circRNAs), a novel class of noncoding RNAs, play a crucial role in the development of cancers, including intrahepatic cholangiocarcinoma (iCCA). Nevertheless, their functions and exact mechanisms in iCCA progression and metastasis are still unclear. Ipatasertib is a highly selective inhibitor of AKT that inhibits tumor growth by blocking the PI3K/AKT pathway. In addition, phosphatase and tensin homolog (PTEN) can also inhibit the activation of the PI3K/AKT pathway, but it is not clear whether the cZNF215-PRDX-PTEN axis plays a role in the antitumor activity of ipatasertib.MethodsWe identified a new circRNA (circZNF215, termed cZNF215) through high-throughput circRNA sequencing (circRNA-seq). In addition, RT‒qPCR, immunoblot assay, RNA pull-down assay, RNA immunoprecipitation (RIP) assay, and fluorescence in situ hybridization assay (FISH) were used to investigate the interaction of cZNF215 with peroxiredoxin 1 (PRDX1). Coimmunoprecipitation (Co-IP) assays and duolink in situ proximity ligation assays (PLAs) were conducted to analyze the effects of cZNF215 on the interaction between PRDX1 and PTEN. Finally, we tested the potential effects of cZNF215 on the antitumor activity of ipatasertib with in vivo experiments.ResultsWe found that cZNF215 expression was obviously upregulated in iCCA tissues with postoperative metastases and was correlated with iCCA metastasis and poor outcome in patients with iCCA. We further revealed that overexpression of cZNF215 promoted iCCA cell growth and metastasis in vitro and in vivo, while cZNF215 knockdown had the opposite effect. Mechanistic studies suggested that cZNF215 competitively interacted with PRDX1, which blocked the association between PRDX1 and PTEN, subsequently leading to oxidation-induced inactivation of the PTEN/AKT pathway and finally contributing to iCCA progression and metastasis. Additionally, we also revealed that silencing cZNF215 in iCCA cells had the potential to enhance the antitumor effect of ipatasertib.ConclusionsOur study demonstrates that cZNF215 facilitates iCCA progression and metastasis by regulating the PTEN/AKT pathway and may serve as a novel prognostic predictor in patients with iCCA.

Redox-stress response resistance (RRR) mediated by hyperoxidation of peroxiredoxin 2 in senescent cells.

Aging is closely related to redox regulation. In our previous work, we proposed a new concept, "redox-stress response capacity (RRC)," and found that the decline in RRC was a dynamic characteristic of aging. However, the mechanism of RRC decline during aging remains unknown. In this study, using the senescent human fibroblast cell model and Caenorhabditis elegans model, we identified that peroxiredoxin 2 (PRDX2), as a hydrogen peroxide (H2O2) sensor, was involved in mediating RRC. PRDX2 knockdown led to a decline of RRC and accelerated senescence in fibroblasts and prdx-2 mutant C. elegans also showed decreased RRC. The mechanism study showed that the decreased sensor activity of PRDX2 was related to the increase in hyperoxidation of PRDX2 in senescent cells. Moreover, the level of PRDX2 hyperoxidation also increased in old C. elegans. Simultaneous overexpression of both PRDX2 and sulfiredoxin (SRX) rescued the reduced RRC and delayed senescence. The increase in PRDX2 hyperoxidation in senescent cells led to a decrease in its sensor activity, resulting in the decreased cellular response to H2O2, which is similar to the mechanism of insulin resistance due to the lower insulin receptor sensitivity. Treatment of young cells with a high level of H2O2 to induce a higher level of PRDX2-SO3 resulted in mimicking the RRC decline in senescent cells, which is also similar to a model of insulin resistance induced by high levels of insulin. All these results thrillingly indicate that there is an insulin-resistance-like phenomenon in senescent cells, we named it redox-stress response resistance, RRR. RRR in senescent cells is an important new discovery that explains RRC decline during aging and reveals the internal relationship between redox regulation and aging from a new perspective.

Disulfide-Bond-Induced Structural Frustration and Dynamic Disorder in a Peroxiredoxin from MAS NMR.

Disulfide bond formation is fundamentally important for protein structure and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive μs time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfill other favorable contacts.