RESUMO
This study aims to investigate the influence of thymol on primordial follicle growth and survival, as well as on collagen fibers and stromal cells density in bovine ovarian tissues cultured in vitro. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the thiol levels and the expression of mRNAs for SOD1, CAT, periredoxin 6 (PRDX6) and GPX1 were also investigated. Ovarian cortical tissues were cultured in α-MEM+ alone or with thymol (400, 800, 1600 or 3200⯵g/mL) for six days. Before and after culture, the tissues were processed for histological analysis to evaluate follicular activation, growth, morphology, ovarian stromal cell density and collagen fibers. The levels of mRNA for SOD1, CAT, GPX1 and PRDX6 were evaluated by real-time PCR. The results show that tissues cultured with thymol (400 and 800⯵g/mL) had increased percentages of normal follicles, when compared to tissues cultured in other treatments. At concentrations of 400 and 800⯵g/mL, thymol maintained the rate of normal follicles similar to the uncultured control. In addition, 400⯵g/mL thymol increased follicle activation, collagen fibers and stromal cell density of when compared to tissues cultured in control medium. The presence of 800⯵g/mL thymol in culture medium increased CAT activity, while 400 or 800⯵g/mL thymol reduced mRNA levels for SOD1, CAT and PRDX6, but did not alter GPX1 expression. In conclusion, 400⯵g/mL thymol increases primordial follicle activation, preserves stromal cells, collagen fibers, and down-regulates expression of mRNA for SOD1, CAT and PRDX6 in cultured bovine ovarian tissues.
Assuntos
Catalase , Colágeno , Folículo Ovariano , RNA Mensageiro , Células Estromais , Timol , Animais , Feminino , Bovinos , Timol/farmacologia , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Folículo Ovariano/efeitos dos fármacos , Catalase/metabolismo , Catalase/genética , Colágeno/metabolismo , Colágeno/genética , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Técnicas de Cultura de Tecidos , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Ferroptosis is a form of regulated cell death induced by iron-dependent accumulation of lipid hydroperoxides. Selenoprotein glutathione peroxidase 4 (GPX4) suppresses ferroptosis by detoxifying lipid hydroperoxides via a catalytic selenocysteine (Sec) residue. Sec, the genetically encoded 21st amino acid, is biosynthesized from a reactive selenium donor on its cognate tRNA[Ser]Sec. It is thought that intracellular selenium must be delivered 'safely' and 'efficiently' by a carrier protein owing to its high reactivity and very low concentrations. Here, we identified peroxiredoxin 6 (PRDX6) as a novel selenoprotein synthesis factor. Loss of PRDX6 decreases the expression of selenoproteins and induces ferroptosis via a reduction in GPX4. Mechanistically, PRDX6 increases the efficiency of intracellular selenium utilization by transferring selenium between proteins within the selenocysteyl-tRNA[Ser]Sec synthesis machinery, leading to efficient synthesis of selenocysteyl-tRNA[Ser]Sec. These findings highlight previously unidentified selenium metabolic systems and provide new insights into ferroptosis.
Assuntos
Ferroptose , Ferro , Peroxirredoxina VI , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Selênio , Ferroptose/efeitos dos fármacos , Selênio/metabolismo , Ferro/metabolismo , Humanos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Animais , Peroxirredoxina VI/metabolismo , Peroxirredoxina VI/genética , Camundongos , Selenoproteínas/metabolismo , Selenocisteína/metabolismo , Aminoacil-RNA de Transferência/metabolismoRESUMO
Peroxiredoxin 6 (Prdx6) repairs peroxidized membranes by reducing oxidized phospholipids, and by replacing oxidized sn-2 fatty acyl groups through hydrolysis/reacylation by its phospholipase A2 (aiPLA2) and lysophosphatidylcholine acyltransferase activities. Prdx6 is highly expressed in the lung, and intact lungs and cells null for Prdx6 or with single-point mutations that inactivate either Prdx6-peroxidase or aiPLA2 activity alone exhibit decreased viability, increased lipid peroxidation, and incomplete repair when exposed to paraquat, hyperoxia, or organic peroxides. Ferroptosis is form of cell death driven by the accumulation of phospholipid hydroperoxides. We studied the role of Prdx6 as a ferroptosis suppressor in the lung. We first compared the expression Prdx6 and glutathione peroxidase 4 (GPx4) and visualized Prdx6 and GPx4 within the lung. Lung Prdx6 mRNA levels were five times higher than GPx4 levels. Both Prdx6 and GPx4 localized to epithelial and endothelial cells. Prdx6 knockout or knockdown sensitized lung endothelial cells to erastin-induced ferroptosis. Cells with genetic inactivation of either aiPLA2 or Prdx6-peroxidase were more sensitive to ferroptosis than WT cells, but less sensitive than KO cells. We then conducted RNA-seq analyses in Prdx6-depleted cells to further explore how the loss of Prdx6 sensitizes lung endothelial cells to ferroptosis. Prdx6 KD upregulated transcriptional signatures associated with selenoamino acid metabolism and mitochondrial function. Accordingly, Prdx6 deficiency blunted mitochondrial function and increased GPx4 abundance whereas GPx4 KD had the opposite effect on Prdx6. Moreover, we detected Prdx6 and GPx4 interactions in intact cells, suggesting that both enzymes cooperate to suppress lipid peroxidation. Notably, Prdx6-depleted cells remained sensitive to erastin-induced ferroptosis despite the compensatory increase in GPx4. These results show that Prdx6 suppresses ferroptosis in lung endothelial cells and that both aiPLA2 and Prdx6-peroxidase contribute to this effect. These results also show that Prdx6 supports mitochondrial function and modulates several coordinated cytoprotective pathways in the pulmonary endothelium.
Assuntos
Células Endoteliais , Ferroptose , Fosfolipases A2 do Grupo VI , Peroxidação de Lipídeos , Pulmão , Peroxirredoxina VI , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Piperazinas , Ferroptose/genética , Peroxirredoxina VI/metabolismo , Peroxirredoxina VI/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Pulmão/metabolismo , Pulmão/patologia , Animais , Células Endoteliais/metabolismo , Camundongos , Humanos , Fosfolipases A2/metabolismo , Fosfolipases A2/genética , Camundongos KnockoutRESUMO
OBJECTIVE: To explore the levels of expression and clinical role of peroxiredoxin 6 (PRDX6) in lung adenocarcinoma. METHODS: This retrospective study used a series of bioinformatics methods to detect the levels of expression of and mutations in the PRDX6 gene in a range of cancers and lung adenocarcinoma. Immunohistochemistry was used to verify the levels of expression of PRDX6 protein in samples of lung adenocarcinoma compared with normal adjacent tissue. The effect of PRDX6 gene knockdown on the in vitro proliferation of a lung adenocarcinoma cell line was measured. Bioinformatics methods were used to determine the diagnostic value and impact on survival of the PRDX6 gene in patients with lung adenocarcinoma. RESULTS: The results showed that the PRDX6 gene was highly expressed in lung adenocarcinoma and there were five mutations at different sites on the gene. PRDX6 promoted the proliferation of the lung adenocarcinoma cell line. The survival duration of lung adenocarcinoma patients with high levels of PRDX6 gene expression was significantly shorter than that of patients with low PRDX6 gene expression. CONCLUSION: PRDX6 is highly expressed in lung adenocarcinoma and higher levels of expression of the PRDX6 gene were associated with a poorer prognosis.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Estudos Retrospectivos , Adenocarcinoma de Pulmão/genética , Linhagem Celular , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologiaRESUMO
The crosstalk between astrocytes and microglia plays a pivotal role in neuroinflammation following ischemic stroke, and phenotypic distribution of these cells can change with the progression of ischemic stroke. Peroxiredoxin (PRDX) 6 phospholipase A2 (iPLA2) activity is involved in the generation of reactive oxygen species(ROS), with ROS driving the activation of microglia and astrocytes; however, its exact function remains unexplored. MJ33, PRDX6D140A mutation was used to block PRDX6-iPLA2 activity in vitro and vivo after ischemic stroke. PRDX6T177A mutation was used to block the phosphorylation of PRDX6 in CTX-TNA2 cell lines. NAC, GSK2795039, Mdivi-1, U0126, and SB202190 were used to block the activity of ROS, NOX2, mitochondrial fission, ERK, and P38, respectively, in CTX-TNA2 cells. In ischemic stroke, PRDX6 is mainly expressed in astrocytes and PRDX6-iPLA2 is involved in the activation of astrocytes and microglia. In co-culture system, Asp140 mutation in PRDX6 of CTX-TNA2 inhibited the polarization of microglia, reduced the production of ROS, suppressed NOX2 activation, and inhibited the Drp1-dependent mitochondrial fission following OGD/R. These effects were further strengthened by the inhibition of ROS production. In subsequent experiments, U0126 and SB202190 inhibited the phosphorylation of PRDX6 at Thr177 and reduced PRDX6-iPLA2 activity. These results suggest that PRDX6-iPLA2 plays an important role in the astrocyte-induced generation of ROS and activation of microglia, which are regulated by the activation of Nox2 and Drp1-dependent mitochondrial fission pathways. Additionally, PRDX6-iPLA2 activity is regulated by MAPKs via the phosphorylation of PRDX6 at Thr177 in astrocytes.
Assuntos
Astrócitos , Butadienos , AVC Isquêmico , Nitrilas , Humanos , Espécies Reativas de Oxigênio/metabolismo , Astrócitos/metabolismo , Microglia/metabolismo , Doenças Neuroinflamatórias , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismoRESUMO
As one of the common plasticizers, di-n-butyl phthalate (DBP) has been using in various daily consumer products worldwide. Since it is easily released from products and exists in the environment for a long time, it has a lasting impact on human health, especially male reproductive health. However, the detailed mechanism of testicular damage from DBP and the protection strategy are still not clear enough. In this study, we found that DBP could induce dose-dependent ferroptosis in testicular tissue. Mechanism dissection indicates that DBP can upregulate SP1 expression, which could directly transcriptionally upregulate PRDX6, a negative regulator of ferroptosis. Overexpression of PRDX6 or adding SP1 agonist curcumin could suppress the DBP-induced ferroptosis on testicular cells. In vivo, rats were given 500 mg/kg/day DBP orally for 3 weeks; elevated levels of ferroptosis were detected in testicular tissue. When the above-mentioned doses of DBP and curcumin at a dose of 300 mg/kg/day were administered intragastrically simultaneously, the testicular ferroptosis induced by DBP was alleviated. Immunohistochemistry and quantitative real-time PCR of testis tissue showed that the expression of PRDX6 was upregulated under the action of DBP and curcumin. These findings suggest a spontaneous self-protection mechanism of testicular tissue from DBP damage by upregulating SP1 and PRDX6. However, it is not strong enough to resist the DBP-induced ferroptosis. Curcumin can strengthen this self-protection mechanism and weaken the level of ferroptosis induced by DBP. This study may help us to develop a novel therapeutic option with curcumin to protect the testicular tissue from ferroptosis and function impairment by DBP.
Assuntos
Curcumina , Ferroptose , Ratos , Masculino , Humanos , Animais , Testículo , Dibutilftalato/toxicidade , Dibutilftalato/metabolismo , Curcumina/farmacologia , Curcumina/metabolismo , Plastificantes/toxicidade , Plastificantes/metabolismo , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismoRESUMO
The continuum of antioxidant response dysregulation in aging/oxidative stress-driven Nlrp3 inflammasome activation-mediated inflammatory response is associated with age-related diseases. Peroxiredoxin (Prdx) 6 is a key antioxidant that provides cytoprotection by regulating redox homeostasis. Herein, using lens epithelial cells (LECs) derived from the targeted inactivation of Prdx6 gene and aging lenses, we present molecular evidence that Prdx6-deficiency causes oxidative-driven Nlrp3 inflammasome activation, resulting in pyroptosis in aging/redox active cells wherein Prdx6 availability offsets the inflammatory process. We observed that Prdx6-/- and aging LECs harboring accumulated reactive oxygen species (ROS) showed augmented activation of Nlrp3 and bioactive inflammatory components, like Caspase-1, IL-1ß, ASC and Gasdermin-D. Similar to lipopolysaccharide treatment, oxidative exposure led to further ROS amplification with increased activation of the Nlrp3 inflammasome pathway. Mechanistically, we found that oxidative stress enhanced Kruppel-like factor 9 (Klf9) expression in aging/Prdx6-/- mLECs, leading to a Klf9-dependent increase in Nlrp3 transcription, while the elimination of ROS by the delivery of Prdx6 or by silencing Klf9 prevented the inflammatory response. Altogether, our data identify the biological significance of Prdx6 as an intrinsic checkpoint for regulating the cellular health of aging or redox active LECs and provide opportunities to develop antioxidant-based therapeutic(s) to prevent oxidative/aging-related diseases linked to aberrant Nlrp3 inflammasome activation.
Assuntos
Antioxidantes , Inflamassomos , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/metabolismo , Inflamassomos/metabolismo , Estresse Oxidativo , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Células Epiteliais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
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-1ß, 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.
Assuntos
Inflamação , Macrófagos , Peroxirredoxina VI , Animais , Camundongos , Citocinas/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Peroxirredoxina VI/genética , Células RAW 264.7 , Transdução de SinaisRESUMO
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with selective degeneration of motor neurons. It has been reported that an increase in the levels of inflammatory cytokines and glial cells such as reactive astrocytes is closely involved in the pathological progression of ALS. Recently, the levels of neuropathic cytotoxic (A1) astrocytes among reactive astrocytes have reportedly increased in the central nervous system of ALS mice, which induce motor neuron degeneration through the production of inflammatory cytokines and secretion of neuropathic factors. Hence, elucidating the induction mechanism of A1 astrocytes in ALS is important to understand the mechanism of disease progression in ALS. In this study, we observed that the expression of peroxiredoxin 6 (PRDX6), a member of the peroxiredoxin family, was markedly upregulated in astrocytes of the lumbar spinal cord of SOD1G93A mice model for ALS. Additionally, when PRDX6 was transiently transfected into the mouse astrocyte cell line C8-D1A and human astrocytoma cell line U-251 MG, the mRNA expression of complement C3 (a marker for A1 astrocyte phenotype) and inflammatory cytokines was increased. Furthermore, the mRNA expression of C3 and inflammatory cytokine was increased in C8-D1A and U-251 MG cells stably expressing PRDX6, and the increased mRNA expression was significantly suppressed by MJ33 (lithium[1-hexadecoxy-3-(2,2,2-trifluoroethoxy) propan-2-yl] methyl phosphate), an inhibitor of the phospholipase A2 activity of PRDX6. Our results suggest that the expression of PRDX6 in astrocytes plays an important role in the induction of A1 astrocytes and expression of inflammatory cytokines in the ALS mice model.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Síndromes Neurotóxicas , Camundongos , Humanos , Animais , Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Doenças Neurodegenerativas/metabolismo , Camundongos Transgênicos , Medula Espinal/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Síndromes Neurotóxicas/metabolismo , RNA Mensageiro/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: PRDX6 gene polymorphism with smoking status may contribute to the etiology of COPD in the Chinese Han population.
Assuntos
População do Leste Asiático , Predisposição Genética para Doença , Doença Pulmonar Obstrutiva Crônica , Humanos , Estudos de Casos e Controles , China/epidemiologia , População do Leste Asiático/etnologia , População do Leste Asiático/genética , Frequência do Gene , Estudos de Associação Genética , Genótipo , Peroxirredoxina VI/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Doença Pulmonar Obstrutiva Crônica/etnologia , Doença Pulmonar Obstrutiva Crônica/genéticaRESUMO
BACKGROUND: Semen cryopreservation is a critical tool for breed improvement and preservation of biodiversity. However, instability of sperm freezability affects its application. The Mediterranean buffalo is one of the river-type buffaloes with the capacity for high milk production. Until now, there is no specific cryopreservation system for Mediterranean buffalo, which influences the promotion of excellent cultivars. To improve the semen freezing extender used in cryopreservation of Mediterranean buffalo, different protein datasets relating to freezability sperm were analyzed by iTRAQ-based proteomics. This study will be beneficial for further understanding the sperm freezability mechanism and developing new cryopreservation strategy for buffalo semen. RESULTS: 2652 quantified proteins were identified, including 248 significantly differentially expressed proteins (DEP). Gene Ontology (GO) analysis indicated that many these were mitochondrial proteins, enriched in the molecular function of phospholipase A2 activity and enzyme binding, and biological processes of regulation of protein kinase A signaling and motile cilium assembly. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 17 significant pathways, including oxidative phosphorylation (OXPHOS). Furthermore, 7 DEPs were verified using parallel reaction monitoring or western blot, which confirmed the accuracy of the iTRAQ data. Peroxiredoxin 6 (PRDX6), which expressed 1.72-fold higher in good freezability ejaculate (GFE) compared to poor freezability ejaculate (PFE) sperms, was selected to explore the function in sperm freezability by adding recombinant PRDX6 protein into the semen freezing extender. The results showed that the motility, mitochondrial function and in vitro fertilization capacity of frozen-thawed sperm were significantly increased, while the oxidation level was significantly decreased when 0.1 mg/L PRDX6 was added compared with blank control. CONCLUSIONS: Above results revealed the metabolic pattern of freezability of Mediterranean buffalo sperms was negatively associated with OXPHOS, and PRDX6 had protective effect on cryo-damage of frozen-thawed sperms.
Assuntos
Búfalos , Preservação do Sêmen , Animais , Masculino , Peroxirredoxina VI/genética , Peroxirredoxina VI/análise , Proteômica , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Espermatozoides/fisiologia , Criopreservação/veterinária , Criopreservação/métodos , Proteínas Recombinantes , Motilidade dos EspermatozoidesRESUMO
1-Cys peroxiredoxin6 (Prdx6) is unique and inducible bifunctional enzyme in the mammalian lungs and plays a role in the progression and inhibition of cancerous cells at different stages. The enzyme possesses two distinct active sites for phospholipase A2 and peroxidase activity. The conserved residues surrounding the peroxidase active site, also called as second shell residues are Glu50, Leu71, Ser72, His79 and Arg155. Since there is no study done about the active site stabilization of the transition state of Prdx6, there are a lot of questions unanswered regarding the Prdx6 peroxidase activity. In order to evaluate the role of second shell conserved residue Glu50, present in close vicinity to peroxidatic active site, we substituted this negatively charged residue with Alanine and Lysine. To explore the effect of mutation on the biophysical parameters, the mutant proteins were compared with Wild-Type by using biochemical, biophysical, and in silico methods. Comparative spectroscopic methods and enzyme activity demonstrate that the Glu50 plays a significant role in maintaining the structure, stability, and function of protein. From the results we conclude that Glu50 significantly controls the structure; stability and may be involved in the active site stabilization of transition state for proper position of diverse peroxides.
Assuntos
Peroxidases , Peroxirredoxina VI , Animais , Peroxirredoxina VI/genética , Peroxirredoxina VI/química , Peroxidases/metabolismo , Fosfolipases A2/metabolismo , Peroxidase/metabolismo , Antioxidantes/química , Mamíferos/metabolismoRESUMO
Peroxiredoxin 6 (Prdx6) is a multifunctional eukaryotic antioxidant enzyme. Mammalian Prdx6 possesses peroxidase activity against a wide range of organic and inorganic hydroperoxides, as well as exhibits phospholipase A2 (aiPLA2) activity, which plays an important role in the reduction of oxidized phospholipids and cell membrane remodeling. Exogenous Prdx6 has recently been shown to be able to penetrate inside the cell. We hypothesized that this entry may be due to the phospholipase activity of Prdx6. Experiments using exogenous Prdx6 in three cell lines (3T3, A549, RAW 264.7) demonstrated that it is the phospholipase activity that promotes its penetration into the cell. Overoxidation of Prdx6 led to a suppression of the peroxidase activity and a 3-to-4-fold growth of aiPLA2, which enhanced the efficiency of its transmembrane transport into the cells by up to 15 times. A mutant form of Prdx6-S32A with an inactivated phospholipase center turned out to be unable to enter the cells in both the reduced and oxidized state of the peroxidase active center. Previously, we have shown that exogenous Prdx6 has a significant radioprotective action. However, the role of phospholipase activity in the radioprotective effects of Prdx6 remained unstudied. Trials with the mutant Prdx6-S32A form, with the use of a total irradiation model in mice, showed a nearly 50% reduction of the radioprotective effect upon aiPLA2 loss. Such a significant decrease in the radioprotective action may be due to the inability of Prdx6-S32A to penetrate animal cells, which prevents its reduction by the natural intracellular reducing agent glutathione S-transferase (πGST) and lowers the efficiency of elimination of peroxides formed from the effect of ionizing radiation. Thus, phospholipase activity may play an important role in the reduction of oxidized Prdx6 and manifestation of its antioxidant properties.
Assuntos
Peroxidase , Peroxirredoxina VI , Camundongos , Animais , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Peroxidase/metabolismo , Fosfolipases A2/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Peroxidases , Mamíferos/metabolismoRESUMO
Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme, a unique member of the peroxiredoxin family, with an important role in antioxidant defense. Moreover, it has also been linked with the biosynthesis of anti-inflammatory and anti-diabetic lipids called fatty acid esters of hydroxy fatty acids (FAHFAs) and many diseases, including cancer, inflammation, and metabolic disorders. Here, we performed metabolomic and lipidomic profiling of subcutaneous adipose tissue from mouse models with genetically modified Prdx6. Deletion of Prdx6 resulted in reduced levels of FAHFAs containing 13-hydroxylinoleic acid (13-HLA). Mutation of Prdx6 C47S impaired the glutathione peroxidase activity and reduced FAHFA levels, while D140A mutation, responsible for phospholipase A2 activity, showed only minor effects. Targeted analysis of oxidized phospholipids and triacylglycerols in adipocytes highlighted a correlation between FAHFA and hydroxy fatty acid production by Prdx6 or glutathione peroxidase 4. FAHFA regioisomer abundance was negatively affected by the Prdx6 deletion, and this effect was more pronounced in longer and more unsaturated FAHFAs. The predicted protein model of Prdx6 suggested that the monomer-dimer transition mechanism might be involved in the repair of longer-chain peroxidized phospholipids bound over two monomers and that the role of Prdx6 in FAHFA synthesis might be restricted to branching positions further from carbon 9. In conclusion, our work linked the peroxidase activity of Prdx6 with the levels of FAHFAs in adipose tissue.
Assuntos
Metabolômica , Peroxirredoxina VI , Animais , Camundongos , Peroxirredoxina VI/genética , Peroxirredoxinas , Adipócitos , Antioxidantes , Ácidos Graxos , FosfolipídeosRESUMO
Hepatic fibrosis is a chronic inflammatory process with hepatic stellate cells (HSCs) activation. Peroxiredoxin 6 (PRDX6), a multifunctional protein, was reported to protect against liver injury induced by ischemia/reperfusion and high-fat diet. However, the effect of PRDX6 on hepatic fibrosis remains unclear. Male Sprague-Dawley rats were treated with carbon tetrachloride (CCl4) for 4-8 weeks to induce hepatic fibrosis. Here, we found that PRDX6 was mainly expressed in hepatocytes and significantly upregulated in CCl4-induced liver fibrosis. To clarify the impact of PRDX6 in hepatic fibrosis, we constructed a PRDX6 knockout (PRDX6-/-) rat model by using CRISPR/Cas9 method. We found that PRDX6 deficiency accelerated CCl4-induced liver fibrosis. Furthermore, we found that PRDX6 knockout promoted α-SMA expression in normal and fibrotic conditions, especially in hepatic fibrosis. PRDX6 knockout significantly upregulated Col1α1 and Col3α1 in fibrotic tissues. To explore the underlying mechanisms, we identified mesencephalic astrocyte-derived neurotrophic factor (MANF), a suppressor for hepatic fibrosis and NF-κB pathway, as an interacting protein of PRDX6. PRDX6 promoted MANF secretion by binding to the C-terminus of MANF, which did not depend on its peroxidase and PLA2 activities. Similarly, MANF increased PRDX6 protein level and promoted its secretion. Additionally, PRDX6 knockout increased p65 level either in cytoplasm or nuclei in HSCs under fibrotic condition. In conclusion, PRDX6 is an effective inhibitor for hepatic fibrosis through a non-enzymic dependent interacting with MANF, which will offer a potential target for hepatic fibrosis therapy.
Assuntos
Células Estreladas do Fígado , Peroxirredoxina VI , Ratos , Masculino , Animais , Células Estreladas do Fígado/metabolismo , Peroxirredoxina VI/genética , Peroxirredoxina VI/farmacologia , Ratos Sprague-Dawley , Fibrose , Cirrose Hepática/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismoRESUMO
Intrahepatic cholangiocarcinoma (ICC) has a poor prognosis. The bifunctional protein peroxiredoxin 6 (PRDX6), which has both calcium-independent phospholipase A2 (iPLA2) and glutathione peroxidase (GPx) activity, participates in the development of multiple tumors. However, the function and clinical significance of PRDX6 in ICC remain unclear. In this study, we characterized PRDX6 in both human ICC and thioacetamide (TAA)-induced rat ICC. We found PRDX6 was significantly increased in ICC tissues, compared with the peritumoral tissues, and PRDX6 expression level was positively correlated with the malignant phenotype in ICC patients. Furthermore, PRDX6 genetic knockout significantly inhibited the tumor progression in rats. By using RNA sequencing analysis, we found 127 upregulated genes and 321 downregulated genes after PRDX6 knockout. In addition, we noticed a significant repression in the Wnt7a/b cascade, which has been shown to play an important role in the occurrence of ICC. We confirmed that gene expressions in the Wnt7a/b cascade were inhibited in ICC tissues after PRDX6 knockout by using qRT-PCR and immunohistochemistry analysis. Collectively, our findings suggest that PRDX6 may promote ICC by regulating the Wnt7a/b pathway, which could be a novel therapeutic target for ICC.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Peroxirredoxina VI/metabolismo , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Peroxirredoxina VI/genética , Ratos , TioacetamidaRESUMO
Background: Systemic lupus erythematosus (SLE) is a complex, multisystem autoimmune disease that is characterized by the production of autoantibodies. Although accumulated evidence suggests that the dysregulation of long non-coding RNAs (lncRNAs) is involved in the pathogenesis of SLE, the genetic contributions of lncRNA coding genes to SLE susceptibility remain largely unknown. Here, we aimed to provide more evidence for the role of lncRNA coding genes to SLE susceptibility. Methods: The genetic association analysis was first adopted from the previous genome-wide association studies (GWAS) and was then validated in an independent cohort. PRDX6-AS1 is located at chr1:173204199-173446294. It spans a region of approximately 240 kb, and 297 single nucleotide polymorphisms (SNPs) were covered by the previous GWAS. Differential expression at the mRNA level was analyzed based on the ArrayExpress Archive database. Results: A total of 33 SNPs were associated with SLE susceptibility, with a P<1.68×10-4. The strongest association signal was detected at rs844649 (P=2.12×10-6), according to the previous GWAS. Combining the results from the GWAS Chinese cohort and our replication cohort, we pursued a meta-analysis approach and found a pronounced genetic association between PRDX6-AS1 rs844649 and SLE susceptibility (pmeta=1.24×10-13, OR 1.50, 95% CI: 1.34-1.67). The mRNA expression of PRDX6 was elevated in peripheral blood cells, peripheral blood mononuclear cells (PBMCs), and multiple cell subpopulations, such as B cells, CD4+ T cells, CD3+ cells, and monocytes in patients with SLE. The PRDX6 protein expression level was also increased in patients with SLE compared with healthy donors. Conclusion: Our study provides new evidence that variants located in lncRNA coding genes are associated with SLE susceptibility.
Assuntos
Lúpus Eritematoso Sistêmico , RNA Longo não Codificante , Humanos , Estudo de Associação Genômica Ampla , Predisposição Genética para Doença , RNA Longo não Codificante/genética , Leucócitos Mononucleares/metabolismo , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , China/epidemiologia , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismoRESUMO
Peroxiredoxin 6 (Prdx6) is an important antioxidant enzyme with multiple functions in the cell. Prdx6 neutralizes a wide range of hydroperoxides, participates in phospholipid metabolism and cell membrane repair, and in transmission of intracellular and intercellular signals. Disruption of normal Prdx6 expression in the cell leads to the development of pathological conditions. Decrease in the Prdx6 concentration leads to increase in oxidative damage to the cell. At the same time, hyperproduction of Prdx6 is associated with increase in antioxidant status, suppression of apoptosis, and carcinogenesis. Currently, mechanisms of carcinogenic action of peroxiredoxins are poorly understood. In this work we established that the 3-4-fold increase in Prdx6 production in mouse embryonic fibroblast 3T3 cells leads to the 4-5-fold decrease in the level of oncosuppressor p53. At the same time, hyperproduction of Prdx6 leads to the increased expression of RELA and HIF1A, which have oncogenic effects. The 3-4-fold increase in intracellular Prdx6 increases intensity of cell proliferation by 20-30%, promotes increase in antioxidant activity by 30-50%, and increases radioresistance of the transfected 3T3 cells by 30-40%. Increase of the level of intranuclear Prdx6 leads to the decrease in expression of the DNA repair genes in response to radiation, indicating decrease in the genomic DNA damage. This work discusses possible molecular mechanisms of p53 suppression during Prdx6 hyperproduction, which could be used in the development of new approaches in cancer therapy.
Assuntos
Antioxidantes , Peroxirredoxina VI , Proteína Supressora de Tumor p53 , Animais , Antioxidantes/metabolismo , Fibroblastos/metabolismo , Camundongos , Estresse Oxidativo , Peroxirredoxina VI/genética , Peroxirredoxina VI/metabolismo , Peroxirredoxinas/metabolismo , Fosfolipídeos , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The human genome has 25 genes coding for selenocysteine (Sec)-containing proteins, whose synthesis is supported by specialized Sec machinery proteins. Here, we carried out an analysis of the co-essentiality network to identify functional partners of selenoproteins and Sec machinery. One outstanding cluster included all seven known Sec machinery proteins and two critical selenoproteins, GPX4 and TXNRD1. Additionally, these nine genes were further positively associated with PRDX6 and negatively with SCD, linking the latter two genes to the essential role of selenium. We analyzed the essentiality scores of gene knockouts in this cluster across one thousand cancer cell lines and found that Sec metabolism genes are strongly selective for a subset of primary tissues, suggesting that certain cancer cell lineages are particularly dependent on selenium. A separate outstanding cluster included selenophosphate synthetase SEPHS1, which was linked to a group of transcription factors, whereas the remaining selenoproteins were linked neither to these clusters nor among themselves. The data suggest that key components of Sec machinery have already been identified and that their primary role is to support the functions of GPX4 and TXNRD1, with further functional links to PRDX6 and SCD.