Peroxiredoxin Research Articles

Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1 (PRDX1) participates in tumor cell proliferation, apoptosis, migration, invasion, and the epithelial-to-mesenchymal transition (EMT). This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide (LPS) and transforming growth factor-beta 1 (TGF-β1). PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial (BEAS-2B) cells, reduced cell apoptosis (p < 0.01), and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase (MMP)2, MMP9, α-smooth muscle actin (α-SMA), N-cadherin, vimentin and twist proteins and inhibiting E-cadherin expression (p < 0.05). PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis (p < 0.05). Knockdown of PRDX1 inhibited cell proliferation, migration, EMT, and collagen synthesis (p < 0.01), reversed LPS-mediated inhibition of cell proliferation and migration, and significantly suppressed LPS-induced EMT and collagen synthesis (p < 0.01). The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.

Integrated bioinformatics and machine learning strategies reveal PRDX6 as the key ferroptosis-associated molecular biosignature of heart failure.

Heart failure (HF) is the leading cause of death and public health problems in the global population. This study aimed to identify and validate ferroptosis-related biomarkers associated with HF in clinical medicine using bioinformatics and machine learning strategies. Weighted co-expression network analysis (WGCNA) was applied to screen the module genes and analyze their biological functions and pathways. Ferroptosis-associated genes (FAG) in HF were determined and then machine learning algorithms were used for screening. Next, multiple external independent microarrays were used to verify molecular biosignature. Simultaneously, CIBERSORT was applied to estimate the immune infiltration landscape. Combined with the results of the WGCNA, 25 FAGs were determined and 6 FAMBs were selected by machine learning strategies. In addition, Peroxiredoxin 6 (PRDX6) was finally selected as the key ferroptosis-associated molecular biological feature based on multiple verifications of independent data sets. From the results of the infiltration and enrichment analysis, we believed that PRDX6, as a protective biomarker related to ferroptosis in HF, may help provide new ideas in the immunotherapy of HF.

The immunohistochemical detection of peroxiredoxin 1 and 2 in canine spontaneous vascular endothelial tumors.

Peroxiredoxin (PRDX) is an antioxidant enzyme family with six isoforms (PRDX1–6). The main function of PRDXs is to decrease cellular oxidative stress by reducing reactive oxygen species, such as hydrogen peroxide, to H2O. Recently, it has been reported that PRDXs are overexpressed in various malignant tumors in humans, and are involved in the development, proliferation, and metastasis of tumors. However, studies on the expression of PRDXs in tumors of animals are limited. Therefore, in the present study, we immunohistochemically investigated the expression of PRDX1 and 2 in spontaneous canine hemangiosarcoma (HSA) and hemangioma (HA), as well as in selected normal tissue and granulation tissue, including newly formed blood vessels. Although there were some exceptions, immunolocalization of PRDX1 and 2 in normal canine tissues was similar to those in humans, rats, or mice. In granulation tissue, angiogenic endothelial cells were strongly positive for PRDX1 and 2, whereas quiescent endothelial cells in mature vessels were negative. Both PRDX1 and 2 were significantly highly expressed in HSA compared to HA. There were no significant differences in the expression of PRDX1 and 2 among the subtypes and primary sites of HSA. These results suggest that PRDX1 and 2 may be involved in the angiogenic phenotypes of endothelial cells in granulation tissue as well as in the behavior in the malignant endothelial tumors.

Peroxiredoxins - Urinary Surveillance Biomarkers in Urothelial Cancer.

Introduction: Urinary bladder cancer ranks the fourth most common cancer in men worldwide. Peroxiredoxins (PRDXs) are antioxidant enzymes that play an important role in cell proliferation and apoptosis. In the present study, we investigated whether PRDX 1 and 2 can be used as a urinary biomarker for surveillance of recurrence in urothelial cancer.Materials and Methods: PRDX1 and PRDX2 expression levels were examined in 119 bladder tumor specimens by immunohistochemistry and in 150 urine samples (case: 100; healthy controls: 50) using ELISA and their association with recurrence and survival of patients was evaluated.Results: Immunohistochemistry on FFPE tissue showed that both PRDX1 and PRDX2 were positive in bladder tumors and expressed in the cytoplasm and membrane of tumor cells. A significant elevation of urinary PRDX1 and PRDX2 concentration was found in bladder cancer patients and recurrent cases compared to the urine of healthy controls and primary bladder cancer patients (p<0.001 & p<0.01) respectively. However, the concentration of both proteins was not found associated with survival.Conclusion: Elevated urinary PRDX1 and PRDX2 in bladder cancer patients was found to be associated with recurrence and the estimation of urinary PRDX1 and PRDX2 during follow-up may help to extend the period between cystoscopies in patient follow-up.

Repurposing Oxiconazole against Colorectal Cancer via PRDX2-mediated Autophagy Arrest

Colorectal cancer (CRC) is one of the most common malignancies worldwide, yet successful treatment still remains a challenge. In this study, we found that oxiconazole (OXI), a broad-spectrum antifungal agent, exhibits certain anti-tumor effect against CRC. Autophagy arrest and subsequent apoptosis are characterized as pivotal events involving OXI-induced growth suppression of CRC cells. Mechanistically, OXI downregulates the protein levels of peroxiredoxin-2 (PRDX2), an antioxidant enzyme, for reactive oxygen species (ROS) detoxication, to initiate autophagy by inactivating the Akt/mTOR pathway and inhibiting RAB7A-mediated fusion of autophagosome and lysosome, which lead to extreme accumulation of autophagosomes and subsequent growth suppression of CRC cells. Consistently, interfering with autophagy or overexpressing PRDX2 significantly impedes OXI-induced growth suppression of CRC cells. Moreover, OXI plus oxaliplatin, a mainstay drug for CRC treatment, achieves an improved anti-tumor effect. Taken together, our findings bring novel mechanistic insights into OXI-induced autophagy arrest and the growth inhibitory effect on CRC cells, and suggest a promisingly therapeutic role of OXI for CRC treatment.

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.

Adaptative Up-Regulation of PRX2 and PRX5 Expression Characterizes Brain from a Mouse Model of Chorea-Acanthocytosis.

The peroxiredoxins (PRXs) constitute a ubiquitous antioxidant. Growing evidence in neurodegenerative disorders such as Parkinson’s disease (PD) or Alzheimer’s disease (AD) has highlighted a crucial role for PRXs against neuro-oxidation. Chorea-acanthocytosis/Vps13A disease (ChAc) is a devastating, life-shortening disorder characterized by acanthocytosis, neurodegeneration and abnormal proteostasis. We recently developed a Vps13a−/− ChAc-mouse model, showing acanthocytosis, neurodegeneration and neuroinflammation which could be restored by LYN inactivation. Here, we show in our Vps13a−/− mice protein oxidation, NRF2 activation and upregulation of downstream cytoprotective systems NQO1, SRXN1 and TRXR in basal ganglia. This was associated with upregulation of PRX2/5 expression compared to wild-type mice. PRX2 expression was age-dependent in both mouse strains, whereas only Vps13a−/− PRX5 expression was increased independent of age. LYN deficiency or nilotinib-mediated LYN inhibition improved autophagy in Vps13a−/− mice. In Vps13a−/−; Lyn−/− basal ganglia, absence of LYN resulted in reduced NRF2 activation and down-regulated expression of PRX2/5, SRXN1 and TRXR. Nilotinib treatment of Vps13a−/− mice reduced basal ganglia oxidation, and plasma PRX5 levels, suggesting plasma PRX5 as a possible ChAc biomarker. Our data support initiation of therapeutic Lyn inhibition as promptly as possible after ChAc diagnosis to minimize development of irreversible neuronal damage during otherwise inevitable ChAc progression.

Comprehensive analysis of the expression levels and prognostic values of PRDX family genes in glioma

Gliomas are a histologically and molecularly heterogeneous group of neoplasms accounting for 80% of malignant primary brain tumors. Growing evidence suggests that production of reactive oxygen species (ROS) is linked to glioma pathogenesis, although it is still unclear whether it is a cause or an effect of this process. Peroxiredoxins (PRDXs), a family of six antioxidant proteins, may promote or inhibit carcinogenesis, depending on the tumor type and stage. The current knowledge on their expression, regulation and functions in glioma is scarce. In this study, a comprehensive analysis of PRDXs expression in distinct glioma subtypes and non-tumor brain tissues was conducted using gene expression data from The Cancer Genome Atlas (TCGA), REpository for Molecular BRAin NeoplasiaDaTa (REMBRANDT), The Chinese Glioma Atlas (CGGA) and Gene Expression Omnibus (GEO) datasets. The association between gene expression and patient survival was investigated. DNA methylation, mutations, copy number alterations of deregulated PRDXs as well as the correlation between gene expression and tumor-infiltrating immune cells were assessed. The analysis revealed overexpression of PRDX1, PRDX4, and PRDX6 in most histological glioma types compared to the non-tumor tissues, while PRDX2, PRDX3 and PRDX5 expression remained unaltered. The expression of PRDX4 and PRDX6 was higher in mesenchymal than proneural and classical glioma subtypes. Moreover, lower expression of PRDX1, PRDX4 and PRDX6 was observed in tumors with a glioma CpG island methylator phenotype (G-CIMP) compared to non-G-CIMP tumors, as well as in isocitrate dehydrogenase (IDH) mutant and 1p/19q co-deleted gliomas compared to the wild-type counterparts. High expression of PRDX1, PRDX4 or PRDX6 correlated with poor survival of glioma patients. PRDX1 and PRDX6 displayed a positive correlation with different immune cell population in low grade gliomas and, to a lesser extent, in glioblastoma. PRDX1 expression exhibited negative correlation with DNA methylation. These results indicate that high expression of PRDX1, PRDX4 and PRDX6 is associated with poor outcome in gliomas.

Zhen-Wu-Tang Induced Mitophagy to Protect Mitochondrial Function in Chronic Glomerulonephritis via PI3K/AKT/mTOR and AMPK Pathways.

Chronic glomerulonephritis (CGN) is one of the major causes of end-stage kidney disease. Zhen-wu-tang (ZWT), as a famous Chinese herbal prescription, is widely used in China for CGN therapy in clinic. However, the mechanism of ZWT in CGN has not been fully understood. The present study explored the therapeutic effect and the underlying mechanism of ZWT on mitochondrial function in cationic bovine serum albumin (C-BSA)-induced CGN model rats and tumor necrosis factor (TNF-α)-damaged mouse podocytes. The renal functions were measured by serum creatinine (Scr) and blood urea nitrogen (BUN). Renal pathological changes and ultrastructure of kidney tissues were evaluated by periodic acid-Schiff (PAS) staining and transmission electron microscopy. The levels of antioxidases, including mitochondrial catalase (CAT), superoxide dismutase 2 (SOD2), and peroxiredoxin 3 (PRDX3), in CGN rats were examined by real-time PCR. The mitochondrial functions of podocytes were measured by ATP concentration, mitochondrial membrane potential (MMP), and mitochondrial ROS (mtROS). For mitophagy level detection, the expressions of mitophagy-related proteins, including LC3, p62, heat shock protein 60 (HSP60), and translocase of outer mitochondrial membrane 20 (TOMM20), were measured by Western blot, as the colocation of LC3 and mitochondrial marker COX IV were evaluated by immunofluorescence. Our results manifested that ZWT ameliorated CGN model rats by a remarkable decrease in Scr and BUN, inhibition of mesangial matrix proliferation, protection against foot processes fusion, and basement membrane thickening. More importantly, ZWT protected against mitochondrial dysfunction by increasing the expressions of CAT, SOD2, and PRDX3 in CGN model rats, increased ATP content and MMP in podocytes, and decreased excessive mtROS. Furthermore, ZWT induced mitophagy in CGN through increasing the expression of LC3, and decreasing p62, HSP60, TOMM20, and ZWT also enhanced the colocation of LC3 to the mitochondria. We found that ZWT inhibited the PI3K/AKT/mTOR pathway, which could be disturbed by PI3K inhibitor LY294002 and agonist insulin-like growth factor 1. Moreover, ZWT reversed the inhibition of the AMPK pathway in CGN. Overall, ZWT ameliorated renal mitochondrial dysfunction probably by inducing mitophagy via the PI3K/AKT/mTOR and AMPK pathways.

Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1

Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron- and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species (ROS) and inducing ferroptosis in activated hepatic stellate cells (HSCs). By using activity-based protein profiling (ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay (CETSA), we show that celastrol directly binds to peroxiredoxins (PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6, through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1 (HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2, PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.

Peroxiredoxin 6 protects irradiated cells from oxidative stress and shapes their senescence-associated cytokine landscape

Cellular senescence is a complex stress response defined as an essentially irreversible cell cycle arrest mediated by the inhibition of cell cycle-specific cyclin dependent kinases. The imbalance in redox homeostasis and oxidative stress have been repeatedly observed as one of the hallmarks of the senescent phenotype. However, a large-scale study investigating protein oxidation and redox signaling in senescent cells in vitro has been lacking. Here we applied a proteome-wide analysis using SILAC-iodoTMT workflow to quantitatively estimate the level of protein sulfhydryl oxidation and proteome level changes in ionizing radiation-induced senescence (IRIS) in hTERT-RPE-1 cells. We observed that senescent cells mobilized the antioxidant system to buffer the increased oxidation stress. Among the antioxidant proteins with increased relative abundance in IRIS, a unique 1-Cys peroxiredoxin family member, peroxiredoxin 6 (PRDX6), was identified as an important contributor to protection against oxidative stress. PRDX6 silencing increased ROS production in senescent cells, decreased their resistance to oxidative stress-induced cell death, and impaired their viability. Subsequent SILAC-iodoTMT and secretome analysis after PRDX6 silencing showed the downregulation of PRDX6 in IRIS affected protein secretory pathways, decreased expression of extracellular matrix proteins, and led to unexpected attenuation of senescence-associated secretory phenotype (SASP). The latter was exemplified by decreased secretion of pro-inflammatory cytokine IL-6 which was also confirmed after treatment with an inhibitor of PRDX6 iPLA2 activity, MJ33. In conclusion, by combining different methodological approaches we discovered a novel role of PRDX6 in senescent cell viability and SASP development. Our results suggest PRDX6 could have a potential as a drug target for senolytic or senomodulatory therapy.

Plasma BDNF and Cytokines Correlated with Protein Biomarkers for Bipolar II Disorder.

We have previously identified five candidate proteins (matrix metallopeptidase 9 (MMP9), phenylalanyl-TRNA synthetase subunit beta (FARSB), peroxiredoxin 2 (PRDX2), carbonic anhydrase 1 (CA-1), and proprotein convertase subtilisin/kexin Type 9 (PCSK9)) as potential biomarkers for bipolar II disorder (BD-II). These candidate proteins have been associated with neuroprotective factors (BDNF) and inflammatory factors (cytokines, C-reactive protein (CRP), and tumor necrosis factor-α (TNF-α)). However, the correlations between these proteins with plasma BDNF and inflammatory factors remain unknown. We recruited a total of 185 patients with BD-II and 186 healthy controls. Plasma levels of candidate proteins, BDNF, cytokines (TNF-α, CRP, and interleukin-8 (IL-8)) were assessed from each participant. The correlations between levels of candidate proteins, BDNF, and cytokines were analyzed. In the BD-II group, we found that the level of FARSB was positively correlated with the BDNF level (r = 0.397, p < 0.001) and IL-8 (r = 0.320, p < 0.001). The CA-1 level positively correlated with IL-8 (r = 0.318, p < 0.001). In the control group, we found that the FARSB level positively correlated with the BDNF level (r = 0.648, p < 0.001). The CA-1 level positively correlated with TNF-α (r = 0.231, p = 0.002), while the MMP-9 level positively correlated with the CRP level (r = 0.227, p = 0.002). Our results may help in clarifying the underlying mechanism of these candidate proteins for BD-II.

Knocking Down Peroxiredoxin 6 Aggravates Cerebral Ischemia–Reperfusion Injury by Enhancing Mitophagy

Cerebral ischemia–reperfusion injury (IRI) is caused by reperfusion following ischemia. Mitophagy is closely related to cerebral IRI. Mitophagy disorder or excess may be harmful and lead to neuronal apoptosis. Peroxiredoxin 6 (PRDX6) is an antioxidant protein and plays an important role in ischemic stroke. However, the relationship between PRDX6 and mitophagy is not clear at present. In order to explore and solve this problem. We have established a middle cerebral artery occlusion (MCAO) model of cerebral ischemia–reperfusion in SD rats and knockdown PRDX6 and PINK1 with lentivirus. Knocking down PRDX6 led to further aggravation of cerebral IRI. Our research found that knockdown PRDX6 increased the expression of mitophagy-related and apoptosis-related proteins. Knocking down PINK1 relieved mitophagy and apoptosis caused by knocking down PRDX6. In conclusion, knockdown of PRDX6 could aggravate cerebral IRI by enhancing PINK1/PARKIN pathway mediated mitophagy, and this effect could increase neuronal apoptosis.

Aloe vera increases collagen fibres in extracellular matrix and mRNA expression of peroxiredoxin-6 in bovine ovarian cortical tissues cultured in vitro.

In vitro culture of ovarian tissue containing primordial follicles is an important tool to study the initiation of follicular populations and to develop efficient culture systems to support in vitro follicle growth. Considering that in vitro culture favours oxidative stress, it is very important to supplement culture medium with antioxidant substances such as Aloe vera extract. This study aims to evaluate the effects of different concentrations of Aloe vera on the distribution of collagen fibres in the extracellular matrix, follicular activation, development and survival in bovine ovarian cortical tissues cultured in vitro, as well as on expression of mRNAs for antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxiredoxin 6 (PRDX6) and glutathione peroxidase 1 (GPX1)]. To this end, ovarian cortical tissues were cultured for 6 days in α-MEM alone or supplemented with different concentrations of Aloe vera extract (1.0, 5.0, 10.0 or 50.0%). After culture, fragments were fixed and processed histologically to evaluate follicular morphology and activation, as well as the extracellular matrix by staining with picrosirius red. The levels of mRNA for SOD, CAT, PRDX6 and GPX1 in cultured ovarian tissues were evaluated by real-time polymerase chain reaction (PCR). Ovarian tissues cultured with 10.0 or 50.0% Aloe vera had higher percentages of collagen fibres than tissues cultured in control medium. A significant increase in developing follicles was observed in ovarian tissues cultured in α-MEM alone or supplemented with 10% Aloe vera when compared with fresh control or tissues cultured with 1.0% Aloe vera. Presence of Aloe vera did not influence the percentage of morphologically normal follicles when compared with control medium. Ovarian tissues cultured with 50.0% Aloe vera had higher percentages of morphologically normal follicles than those cultured with 10.0% Aloe vera. Furthermore, 10% Aloe vera significantly increased mRNA levels for PRDX6. In conclusion, 10.0% Aloe vera improves extracellular matrix distribution in cultured tissues and increases the expression of mRNA for PRDX6 after 6 days in vitro.

PRDX-1 Supports the Survival and Antitumor Activity of Primary and CAR-Modified NK Cells under Oxidative Stress.

Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Effect of 2-Cys Peroxiredoxins Inhibition on Redox Modifications of Bull Sperm Proteins.

Sperm peroxiredoxins (PRDXs) are moonlighting proteins which, in addition to their antioxidant activity, also act as redox signal transducers through PRDX-induced oxidative post-translational modifications of proteins (oxPTMs). Despite extensive knowledge on the antioxidant activity of PRDXs, the mechanisms related to PRDX-mediated oxPTMs are poorly understood. The present study aimed to investigate the effect of bull sperm 2-Cys PRDX inhibition by Conoidin A on changes in oxPTM levels under control and oxidative stress conditions. The results showed that a group of sperm mitochondrial (LDHAL6B, CS, ACO2, SDHA, ACAPM) and actin cytoskeleton proteins (CAPZB, ALDOA, CCIN) is oxidized due to the action of 2-Cys PRDXs under control conditions. In turn, under oxidative stress conditions, 2-Cys PRDX activity seems to be focused on antioxidant function protecting glycolytic, TCA pathway, and respiratory chain enzymes; chaperones; and sperm axonemal tubulins from oxidative damage. Interestingly, the inhibition of PRDX resulted in oxidation of a group of rate-limiting glycolytic proteins, which is known to trigger the switching of glucose metabolism from glycolysis to pentose phosphate pathway (PPP). The obtained results are expected to broaden the knowledge of the potential role of bull sperm 2-Cys in both redox signal transmission and antioxidant activity.

Role of the Redox State of Human Peroxiredoxin-5 on Its TLR4-Activating DAMP Function

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

The BMSC-derived exosomal lncRNA Mir9-3hg suppresses cardiomyocyte ferroptosis in ischemia-reperfusion mice via the Pum2/PRDX6 axis

Background and aimsThe exosomal long noncoding RNAs (lncRNAs) have been reported to have cardioprotective effects on ischemia-reperfusion (I/R) injury by hindering ferroptosis, but the role of lncRNA Mir9-3 host gene (Mir9-3hg) in cardiac I/R injury remains unclear. Methods and resultsExosomes were extracted from mouse bone marrow mesenchymal stem cells (BMSCs) and identified by detecting the exosome specific marker levels, and the results showed that Mir9-3hg was highly expressed in BMSCs-Exo. Hypoxia/reoxygenation (H/R)-treated HL-1 mouse cardiomyocytes were incubated with exosomes extracted from BMSCs transfected with Mir9-3hg siRNA. BMSCs-Exo incubation observably facilitated cell proliferation, increased glutathione (GSH) content, and reduced iron ion concentration, reactive oxygen species (ROS) level and ferroptosis marker protein levels in H/R-treated cells, while interfering Mir9-3hg reversed these effects. RNA binding protein immunoprecipitation assay was found that Mir9-3hg bound with pumilio RNA binding family member 2 (Pum2) protein and downregulated Pum2 expression. Silence of Pum2 reversed the effects of Mir9-3hg inhibition on cell functions. Chromatin immunoprecipitation assay was revealed that Pum2 bound with peroxiredoxin 6 (PRDX6) promoter and restrained PRDX6 expression. Silence of PRDX6 reversed the improved effects of Pum2 downregulation on cell functions. Additionally, BMSCs-Exo treatment ameliorated cardiac function in I/R-treated mice by inhibiting cardiomyocyte ferroptosis. ConclusionsBMSCs-Exo treatment attenuates I/R-induced cardiac injury by inhibiting cardiomyocyte ferroptosis through modulating the Pum2/PRDX6 axis, thereby ameliorating cardiac function.

Peroxiredoxin 1 is essential for natamycin-triggered apoptosis and protective autophagy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide and lacks effective treatment. Herein, we found that the antifungal Natamycin (NAT) exhibits antitumor activity by inducing apoptosis both in vitro and in vivo. Mechanistically, NAT downregulates the expression of Peroxiredoxin 1 (PRDX1) by promoting ubiquitination-mediated degradation, thereby leading to increased reactive oxygen species (ROS) accumulation and subsequent apoptosis. Exogenous overexpression of PRDX1 or N-acetyl-l-cysteine (NAC) pretreatment abrogates NAT-induced cytotoxicity in PLC/PRF/5 and Huh7 cells, suggesting the vital role of ROS in the antitumor properties of NAT. Of note, downregulation of PRDX1 decreases the phosphorylation of AKT, thereby inducing cytoprotective autophagy and combinational use of NAT and chloroquine (CQ) achieves better anti-tumor efficacy. Moreover, NAT acts synergistically with sorafenib (SOR) in HCC suppression. Collectively, our study provides an important molecular basis for NAT-induced cell death and suggests that the antifungal NAT holds the potential to be repurposed as an anticancer drug for HCC treatment.

Redox-sensitive cyclophilin A elicits chemoresistance through realigning cellular oxidative status in colorectal cancer.

Cancer cells utilize rapidly elevated cellular antioxidant programs to accommodate chemotherapy-induced oxidative stress; however, the underlying mechanism remains largely unexplored. Here we screen redox-sensitive effectors as potential therapeutic targets for colorectal cancer (CRC) treatment and find that cyclophilin A (CypA) is a compelling candidate. Our results show that CypA forms an intramolecular disulfide bond between Cys115 and Cys161 upon oxidative stress and the oxidized cysteines in CypA are recycled to a reduced state by peroxiredoxin-2 (PRDX2). Furthermore, CypA reduces cellular reactive oxygen species levels and increases CRC cell survival under insults of H2O2 and chemotherapeutics through a CypA-PRDX2-mediated antioxidant apparatus. Notably, CypA is upregulated in chemoresistant CRC samples, which predicts poor prognosis. Moreover, targeting CypA by cyclosporine A exhibits promising efficacy against chemoresistant CRC when combined with chemotherapeutics. Collectively, our findings highlight CypA as a component of cellular noncanonical antioxidant defense and as a potential druggable therapeutic target to ameliorate CRC chemoresistance.