ER Oxidative Stress Promotes Glutathione-Dependent Oxidation of Collagen-1A1 and Promotes Lung Fibroblast Activation.

Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in collagen 1A1 S-glutathionylation (COL1A1-SSG) in lung tissues from IPF subjects compared to control subjects in association with increases in ER oxidoreductin 1 (ERO1A) and enhanced oxidation of ER-localized peroxiredoxin 4 (PRDX4) reflecting an increased oxidative environment of the endoplasmic reticulum (ER). Human lung fibroblasts exposed to transforming growth factor beta 1 (TGFB1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 levels and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared to COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly, and increased expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling due to increased resistance to collagenase-mediated degradation and fibroblast activation.

Peroxiredoxin-4 supplementation modulates the immune response, shapes the intestinal microbiome, and enhances AHPND resistance in Penaeus vannamei.

Peroxiredoxin-4 from Penaeus vannamei (LvPrx4) is considered a damage-associated molecular pattern (DAMP) that can activate the expression of immune-related genes through the Toll pathway. We previously demonstrated that the recombinant LvPrx4 (rLvPrx4) can enhance shrimp resistance against Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (VPAHPND), which causes great production losses in shrimp farming. Herein, we showed that the rLvPrx4 had a thermal tolerance of around 60 °C and that the ionic strength had no noticeable effect on its activity. We discovered that feeding a diet containing rLvPrx4 to shrimp for three weeks increased the expression of the immune-related genes LvPEN4 and LvVago5. Furthermore, pre-treatment with rLvPrx4 feeding could significantly prolong shrimp survival following the VPAHPND challenge. The shrimp intestinal microbiome was then characterized using PCR amplification of the 16S rRNA gene and Illumina sequencing. Three weeks of rLvPrx4 supplementation altered the bacterial community structure (beta diversity) and revealed the induction of differentially abundant families, including Cryomorphaceae, Flavobacteriaceae, Pirellulaceae, Rhodobacteraceae, and Verrucomicrobiaceae, in the rLvPrx4 group. Metagenomic predictions indicated that some amino acid metabolism pathways, such as arginine and proline metabolism, and genetic information processing were significantly elevated in the rLvPrx4 group compared to the control group. This study is the first to describe the potential use of rLvPrx4 supplementation to enhance shrimp resistance to VPAHPND and alter the composition of a beneficial bacterial community in shrimp, making rLvPrx4 a promising feed supplement as an alternative to antibiotics for controlling VPAHPND infection in shrimp aquaculture.

Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii.

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.

Oxidation of peroxiredoxin-4 induces oligomerization and promotes interaction with proteins governing protein folding and endoplasmic reticulum stress.

Peroxiredoxins (PRDXs) catalyze the reduction of hydrogen peroxide (H2O2). PRDX4 is the only peroxiredoxin located within the endoplasmic reticulum (ER) and is the most highly expressed H2O2 scavenger in the ER. PRDX4 has emerged as an important player in numerous diseases, such as fibrosis and metabolic syndromes, and its overoxidation is a potential indicator of ER redox stress. It is unclear how overoxidation of PRDX4 governs its oligomerization state and interacting partners. Herein, we addressed these questions via nonreducing Western blots, mass spectrometry, and site-directed mutagenesis. We report that the oxidation of PRDX4 in lung epithelial cells treated with tertbutyl hydroperoxide caused a shift of PRDX4 from monomer/dimer to high molecular weight (HMW) species, which contain PRDX4 modified with sulfonic acid residues (PRDX4-SO3), as well as of a complement of ER-associated proteins, including protein disulfide isomerases important in protein folding, thioredoxin domain-containing protein 5, and heat shock protein A5, a key regulator of the ER stress response. Mutation of any of the four cysteines in PRDX4 altered the HMW species in response to tertbutyl hydroperoxide as well as the secretion of PRDX4. We also demonstrate that the expression of ER oxidoreductase 1 alpha, which generates H2O2 in the ER, increased PRDX4 HMW formation and secretion. These results suggest a link between SO3 modification in the formation of HMW PRDX4 complexes in cells, whereas the association of key regulators of ER homeostasis with HMW oxidized PRDX4 point to a putative role of PRDX4 in regulating ER stress responses.

Peroxiredoxin 4 secreted by cumulus cells ameliorates the maturation of oocytes in vitro.

BACKGROUND: Peroxiredoxin 4 (Prdx4) in the endoplasmic reticulum (ER) is the only secretory member of the antioxidant Prdx family. Our previous studies demonstrated that Prdx4 in cumulus cells (CCs) ameliorated the maturation of oocytes in vitro and enhanced oocyte developmental competence by preventing CCs apoptosis caused by oxidative stress (OS) through gap junctions. In this study, we aimed to determine whether Prdx4 released by CCs can repair meiotic defects in mouse oocytes by co-culturing immature (germinal vesicle) oocytes with CCs from mature oocytes in the absence of gap junctions. RESULTS: The OS-induced meiotic defects in mouse oocytes were impeded by co-culture with CCs, as evidenced by the increased first polar body (PB1) extrusion rate and decreased ROS level. CCs increased Prdx4 expression and lowered IRE1α, Bip expression in H2O2-treated oocytes. After knockdown of Prdx4 expression in CCs, the rate of PB1 extrusion in the oocytes was significantly reduced to the level detected in H2O2 group, and ER stress was not alleviated. CO-IP and immunofluorescence co-localization experiments demonstrated that Prdx4 interacted with PDIA6 in the oocytes and the Pearson's R value was 0.69 calculated using ImageJ. CONCLUSIONS: Cumulus cells can promote the maturation of oocytes in vitro by secreting Prdx4 in a paracrine manner and serve as a promising therapeutic antioxidant for improving the quality of oocytes, especially aging oocytes, in clinical in vitro maturation (IVM).

Peroxiredoxin 4, a new favorable regulator, can protect oocytes against oxidative stress damage during in vitro maturation.

BACKGROUND: Finding an effective regulator to avoid harmful effects caused by excessive reactive oxygen species (ROS) is a bottleneck during oocyte in vitro maturation (IVM). Previously, we found that peroxiredoxin 4 (Prdx4) expression is significantly higher in mature cumulus cell-oocyte complexes (COCs) than in immature COCs. Prdx4 belongs to the antioxidant enzyme family and can catalyze the reduction of H2O2. RESULTS: In this study, we established an oxidative stress model with mouse COCs cultured in vitro. Treatment with H2O2 decreased cumulus expansion indexes and oocyte maturation in a concentration-dependent manner, indicating follicular development dysplasia. Infection with a Prdx4-overexpressing adenovirus significantly attenuated H2O2-induced changes, exhibiting effects similar to those of the intracellular ROS scavenger tiron (the positive control). Furthermore, the results confirmed that the protective effect of Prdx4 on oocyte maturation may be due to reductions in ROS levels and apoptosis. However, when the gap junctions between cumulus cells (CCs) and oocytes were destroyed, Prdx4 overexpression did not exert antiapoptotic effects. The expression levels of the gap junction marker protein CX43 were significantly recovered in the Prdx4-overexpressing group. CONCLUSIONS: These results demonstrate that Prdx4 in CCs may be a new favorable regulator that improves in vitro-matured oocyte quality and enhances oocyte developmental competence by preventing CC apoptosis caused by oxidative stress through gap junctions. The findings expand the body of knowledge regarding follicle development, and the identification of Prdx4 as a new favorable regulator will aid in immature oocyte IVM.

Consequences of a peroxiredoxin 4 (Prdx4) deficiency on learning and memory in mice.

Peroxiredoxin 4 (Prdx4) is responsible for the oxidative folding of new proteins that are synthesized in the endoplasmic reticulum (ER). It has recently been suggested that increased ER stress is associated with neurodegenerative diseases, including Alzheimer's disease. Prdx4 is widely distributed throughout the brain, and is also expressed in hippocampal neurons and oligodendrocytes, suggesting that it is associated with learning and memory. We previously established Prdx4-knockout (KO) mice but did not examine the behavioral phenotypes. In the present study, we report on the learning and memory abilities of Prdx4-KO mice based on Morris water maze and the Y-maze tests. The findings indicate that Prdx4-KO mice showed a lower spatial memory ability in both tests. In contrast, the results of the open field test indicated that locomotor activity is significantly increased in Prdx4-KO mice. We then performed mRNA analyses of the brains of Prdx4-KO mice and found an increased expression of genes related to the ER-associated degradation (ERAD) mechanism, which is an important protein quality control system for the maintenance of ER homeostasis. Finally, proteomic analyses of the brains of Prdx4-KO mice showed an aberrant expression in the proteins, which have been suggested to be related to calcium homeostasis and synaptogenesis in neurons. Our collective results suggest that the Prdx4 ablation perturbs oxidative protein folding in the ER, thus leading to aberrant ER homeostasis in neuronal cells, ultimately leading to impaired spatial memory formation.

Modulation of vascular integrity and neuroinflammation by peroxiredoxin 4 following cerebral ischemia-reperfusion injury.

Ischemic stroke is a leading cause of morbidity and mortality worldwide, with oxidative stress playing a key role in the injury mechanism of thrombolytic therapy. There is increasing evidence that oxidative stress damages endothelial cells (ECs), degrades tight junction proteins (TJs), and contributes to increased blood-brain barrier (BBB) permeability. It has been demonstrated that the breakdown of BBB could increase the risk of intracerebral hemorrhagic transformation in ischemic stroke. And an episode of cerebral ischemia/reperfusion (I/R) also initiates oxidative stress-mediated inflammatory processes in ECs, which further promotes BBB disruption and the progression of brain injury. Previous studies have revealed that antioxidants could inhibit ROS generation and attenuate BBB disruption after cerebral I/R. Peroxiredoxin 4 (Prx4) is a member of the antioxidant enzymes family (Prx1-6) and has been characterized to be an efficient H2O2 scavenger. It should be noted that Prx4 may be directly involved in the protection of ECs from the effects of ROS and function in ECs as a membrane-associated peroxidase. This paper reviewed the implication of Prx4 on vascular integrity and neuroinflammation following a cerebral I/R injury.

Peroxiredoxin 4 inhibits insulin-induced adipogenesis through regulation of ER stress in 3T3-L1 cells.

Obesity was originally considered a disease endemic to developed countries but has since emerged as a global health problem. Obesity is characterized by abnormal or excessive lipid accumulation (World Health Organization, WHO) resulting from pre-adipocyte differentiation (adipogenesis). The endoplasmic reticulum (ER) produces proteins and cholesterol and shuttles these compounds to their target sites. Many studies have implicated ER stress, indicative of ER dysfunction, in adipogenesis. Reactive oxygen species (ROS) are also known to be involved in pre-adipocyte differentiation. Prx4 specific to the ER lumen exhibits ROS scavenging activity, and we thereby focused on ER-specific Prx4 in tracking changes in adipocyte differentiation and lipid accumulation. Overexpression of Prx4 reduced ER stress and suppressed lipid accumulation by regulating adipogenic gene expression during adipogenesis. Our results demonstrate that Prx4 inhibits ER stress, lowers ROS levels, and attenuates pre-adipocyte differentiation. These findings suggested enhancing the activity of Prx4 may be helpful in the treatment of obesity; the data also support the development of new therapeutic approaches to obesity and obesity-related metabolic disorders.

Galectin-3 inhibition attenuates doxorubicin-induced cardiac dysfunction by upregulating the expression of peroxiredoxin-4.

Doxorubicin (DOX) is a highly efficient chemotherapeutic drug limited by its cardiotoxicity. Galectin-3 (Gal-3) overexpression is associated with several cardiovascular diseases. In this study, the in vivo models of DOX-treated rats and the in vitro model of DOX-treated H9C2 cells were used. DOX induced cardiac injury and dysfunction accompanied with the upregulation of Gal-3 at the end of the experiment, while inhibition of Gal-3 with modified citrus pectin (MCP) exhibited a dramatic improvement in cardiac function of the DOX-treated rats, as manifested by increased left ventricular systolic pressure and ±dp/dtmax and decreased left ventricular end-diastolic pressure. The plasma levels of myocardial injury markers such as lactate dehydrogenase, creatine kinase, creatine kinase-MB, and cardiac troponin I were decreased after MCP treatment. In parallel, MCP attenuated myocardial tissue markers of oxidative stress such as hydrogen peroxide and malondialdehyde restored the activities of superoxide dismutase, catalase, and glutathione peroxidase and upregulated antioxidant peroxiredoxin-4 (Prx-4). To further verify the role of Prx-4, it was downregulated by siRNA-mediated knockdown in H9C2 cells. MCP could not reverse DOX-induced oxidative stress in Prx-4-knock-down cells. In conclusion, Gal-3 mediated DOX-induced cardiotoxicity and Gal-3 inhibition attenuated DOX-induced cardiac dysfunction by upregulating the expression of Prx-4 to reduce myocardial oxidative stress.

[Peroxiredoxin 4 protects the testis from heat stress].

OBJECTIVE: To explore the protective effect of Peroxiredoxin 4 (PRDX4) on the testes undergoing heat stress in PRDX4 knockout mice. METHODS: Twenty-four C57BL/6 mice underwent CRISPR/Cas9-mediated total knockout of the PRDX4 gene and another 24 wild-type mice were used as controls. At 9 weeks of age, the rats were subjected to 15-minute testicular heat stress in 43℃ water once a day for 3 days, or in 25℃ water as the control. Before and at 1 day and 5 weeks after treatment, 4 from each group were sacrificed respectively and their testes harvested for observation of histological changes by HE staining, detection of the apoptosis of spermatogenic cells by TUNEL and determination of the expression of PRDX4 by Western blot and those of the oxidative stress factors hydroxynonenal (HNE) and 8-OHdG by immunohistochemistry. RESULTS: No statistically significant differences were observed in testicular histology, the apoptosis rate of spermatogenic cells, and the expressions of HNE and 8-OHdG between the PRDX4 knockout mice and wild-type controls (P > 0.05). After 1-day 43℃ heat stress, the PRDX4 knockout mice showed a significantly increased apoptosis rate of spermatogenic cells as compared with the baseline (ï¼»38.65 ± 2.57ï¼½% vs ï¼»0.46 ± 0.06ï¼½%, P < 0.01), and so did the wild-type controls (ï¼»13.21 ± 1.43ï¼½% vs ï¼»0.33 ± 0.01ï¼½%, P < 0.01), higher in the PRDX4 knockout than in the wild-type control group even at 5 weeks after heat stress (ï¼»3.09 ± 0.16ï¼½% vs ï¼»1.45 ± 0.11ï¼½%, P < 0.01). The PRDX4 knockout mice also exhibited a markedly upregulated expression of 8-OHdG (38.25 ± 1.19 vs 19.54 ± 1.13, P < 0.01), and so did the wild-type controls (24.30 ± 1.65 vs 18.22 ± 1.18, P < 0.01), higher in the PRDX4 knockout than in the wild-type control group even at 5 weeks after heat stress (25.40 ± 1.57 vs 23.25 ± 1.48, P < 0.01). The expression of HNE, however, showed no statistically significant difference before and at 1 day after 43℃ heat stress either in the PRDX4 knockout mice or in the wild-type controls (P > 0.05), though remarkably higher in the former than in the latter group at 5 weeks after treatment (28.57 ± 0.56 vs 19.00 ± 1.35, P < 0.01). The expression of 8-OHdG was also higher in the PRDX4 knockout than in the wild-type control group at 5 weeks, but with no statistically significant difference (P > 0.05). CONCLUSIONS: PRDX4 can effectively protect the testis from heat stress and promote the restoration of its spermatogenic function.

Cloning and functional characterization of rockfish peroxiredoxin 4 homolog with its innate immune responses.

The fourth member of the typical 2-cysteine peroxiredoxin (Prx4) is a well-known antioxidant enzyme, which reduces different peroxides in their catalytic process. The present study reports the identification of the rockfish Sebastes schlegelii Prx4 (SsPrx4) at a genomic level, as well as the characterization of its structural and functional features. SsPrx4 harbors a complete ORF of 786 bp encoding a polypeptide (29 kDa) of 262 amino acids (aa) with an isoelectric point of 6.2. Thioredoxin 2 domain was prominent in the SsPrx4 sequence, which has a signal peptide (31 bp) at the N-terminus. Hence, the SsPrx4 may be functionally active in the cytoplasm of rockfish cells. Moreover, two VCP motifs and three catalytic triad residues (112T, 115C, 191R) were identified in the SsPrx4 protein sequence. A peroxidatic cysteine (115CP) and resolving cysteines (236CR) were detected at the VCP motifs. The rockfish Prx4 genome consists of seven exons, which are similar to the architecture of other Prx4 orthologs. The deduced amino acid sequence of SsPrx4 shares a relatively high amino acid sequence identity (91.6%) and close evolutionary relationship with Miichthys miiuy and Stegastes partitus Prx4. The potential for scavenging extracellular H2O2 was evidenced by the purified recombinant SsPrx4 protein (rSsPrx4) in vitro system. Moreover, rSsPrx4 may protect the plasmid DNA in a metal-catalyzed oxidation system and catalyze the reduction of an insulin disulfide bond. Quantitative real-time PCR revealed that SsPrx4 mRNA was ubiquitously expressed in fourteen different tissues, with the highest expression observed in the liver followed by the ovary, and kidney tissues. Transcriptional modulations were observed in liver and spleen tissues of rockfish after injecting them with bacterial stimuli, including Streptococcus iniae, LPS, and a viral mimic of poly I:C. Together, the results suggest that SsPrx4 may play an important role in both the antioxidant and innate immune defense of black rockfish. These findings provide structural and functional insights into the SsPrx4 of the teleost.

Peroxiredoxin 4 ameliorates amyloid beta oligomer-mediated apoptosis by inhibiting ER-stress in HT-22 hippocampal neuron cells.

Alzheimer's disease (AD) is a neurodegenerative disorder caused by amyloid beta oligomers (AßO), which induce cell death by triggering oxidative stress and endoplasmic reticulum (ER) stress. Oxidative stress is regulated by antioxidant enzymes, including peroxiredoxins. Peroxiredoxins (Prx) are classified into six subtypes, based on their localization and cysteine residues, and protect cells by scavenging hydrogen peroxide (H2O2). Peroxiredoxin 4 (Prx4) is unique in being localized to the ER; however, whether Prx4 protects neuronal cells from AßO-induced toxicity remains unclear, although Prx4 expression is upregulated in AßO-induced oxidative stress and ER stress. In this study, we established HT-22 cells in which Prx4 was either overexpressed or silenced to investigate its role in AßO-induced toxicity. AßO-stimulation of HT-22 cells with overexpressed Prx4 caused decreases in both AßO-induced ROS and ER stress (followed by ER expansion). In contrast, AßO stimulation caused increases in both ROS and ER stress that were notably higher in HT-22 cells with silenced Prx4 expression than in HT-22 cells. Consequently, Prx4 overexpression decreased apoptotic cell death and ameliorated the AßO-induced increase in intracellular Ca2+. Therefore, we conclude that Prx4 has a protective effect against AßO-mediated oxidative stress, ER stress, and neuronal cell death. Furthermore, these results suggest that Prx4 may be a target for preventing AßO toxicity in AD. Graphical abstract .

The overexpression of peroxiredoxin-4 affects the progression of idiopathic pulmonary fibrosis.

BACKGROUND: Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is life-threatening. Several serum biomarkers, such as Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), are clinically used for evaluating AE-IPF, but these biomarkers are not adequate for establishing an early and accurate diagnosis of AE-IPF. Recently, the protective roles of the members of the peroxiredoxin (PRDX) family have been reported in IPF; however, the role of PRDX4 in AE-IPF is unclear. METHODS: Serum levels of PRDX4 protein, KL-6, SP-D and lactate dehydrogenase (LDH) in 51 patients with stable IPF (S-IPF), 38 patients with AE-IPF and 15 healthy volunteers were retrospectively assessed using enzyme-linked immunosorbent assay. Moreover, as an animal model of pulmonary fibrosis, wild-type (WT) and PRDX4-transgenic (Tg) mice were intratracheally administered with bleomycin (BLM, 2 mg/kg), and fibrotic and inflammatory changes in lungs were evaluated 3 weeks after the intratracheal administration. RESULTS: Serum levels of PRDX4 protein, KL-6, SP-D and LDH in patients with S-IPF and AE-IPF were significantly higher than those in healthy volunteers, and those in AE-IPF patients were the highest among the three groups. Using receiver operating characteristic curves, area under the curve values of serum PRDX4 protein, KL-6, SP-D, and LDH for detecting AE-IPF were 0.873, 0.698, 0.675, and 0.906, respectively. BLM-treated Tg mice demonstrated aggravated histopathological findings and poor prognosis compared with BLM-treated WT mice. Moreover, PRDX4 expression was observed in alveolar macrophages and lung epithelial cells of BLM-treated Tg mice. CONCLUSIONS: PRDX4 is associated with the aggravation of inflammatory changes and fibrosis in the pathogenesis of IPF, and serum PRDX4 may be useful in clinical practice of IPF patients.

Increased circulating peroxiredoxin-4 in sepsis model rats involves secretion from hepatocytes and is mitigated by GYY4137.

Circulating peroxiredoxin-4 (Prx4) is suggested as a prognosis marker as well as a regulator of many diseases. We aimed to examine 1) whether Prx4 is secreted from the liver in an animal model of sepsis and 2) effects of GYY4137, a hydrogen sulfide donor molecule, on septic liver injury as well as the hepatic secretion of Prx4. Rats (Wistar, male, 6 weeks old) were administered lipopolysaccharide (LPS, 15 mg/kg body weight, i.p.) with or without pre-administration of GYY4137 (50 mg/kg body weight, i.p.) and sacrificed 24 h after LPS administration. Hematoxylin-eosin and Elastica Masson-Goldner stains were used to evaluate hepatic injuries. Cytokine expression levels were determined by qPCR, and the levels of Prx4 in the serum and liver were determined by immunoblotting. Hepatocytes were isolated from rat liver, and the levels of Prx4 in the medium as well as the cells were determined 24 h after the administrations of LPS (1 µg/ml), tumor necrosis factor-α (TNFα, 50 ng/ml), or interleukin-1ß (IL-1ß, 10 ng/ml), with or without GYY4137 (300 µM). Hepatic inflammation and damage in LPS-administered rats were suppressed by GYY4137. An increase in plasma Prx4 level caused by LPS was observed, but the increase was attenuated by pre-administration of GYY4137. Prx4 was secreted from isolated hepatocytes after stimulation with LPS, TNFα, or IL-1ß. GYY4137 attenuated the IL-1ß-induced Prx4 secretion from hepatocytes. Secretion from hepatocytes is likely involved in the increase in circulating Prx4 during sepsis. GYY4137 attenuates not only hepatic injury but also Prx4 secretion.

Recombinant protein transduction domain-Cu/Zn superoxide dismutase alleviates bone cancer pain via peroxiredoxin 4 modulation and antioxidation.

Bone cancer pain (BCP) is a serious chronic clinical condition and reactive oxygen species (ROS) were considered to be involved in its development and persistency. Normally, superoxide dismutase (SOD) converts superoxide anions to hydrogen peroxide (H2O2) and H2O2 is then naturalized to be water by peroxiredoxin 4. We reported previously that recombinant protein transduction domain (PTD)-Cu/Zn SOD effectively scavenged excessive ROS and prevented cardiomyocytes from hypoxia-reoxygenation damage. However, whether PTD-Cu/Zn SOD would prevent BCP development is unknown. In the current study, we found that an implanted carcinoma in the rat tibia induced remarkable hyperalgesia, increased H2O2 levels and decreased SOD and peroxiredoxin 4 levels. After administration of recombinant PTD-Cu/Zn SOD to these tumor-burden rats, their hyperalgesia was significantly attenuated and peroxiredoxin 4 expression was significantly increased. In addition, an increased expression of N-methyl-d-aspartic acid (NMDA) receptors and a decreased expression of γ-aminobutyric acid (GABA) receptors in this cancer pain were prevented by PTD-Cu/Zn SOD administration or peroxiredoxin 4 overexpression. Our data suggested that reactive oxygen species, at least in part, play a role in cancer metastatic pain development and persistency which can be attenuated by the adminstration of recombinant PTD-Cu/Zn SOD via the peroxiredoxin 4 modulation from oxidative stress.

Early and enduring: Targeting the endothelium for blood-brain barrier protection.

The disruption of the blood-brain barrier marks a pivotal early pathological event in ischemic stroke that significantly contributes to subsequent permanent damage. Here we delve into the ramifications of a study conducted by Xu and colleagues, which underscores the essential role of the protein peroxiredoxin-4 in cerebrovascular endothelial cells. Peroxiredoxin-4 was shown to preserve blood-brain barrier integrity during the early stages after cerebral ischemia and reperfusion, ultimately leading to improved long-term outcomes.

Peroxiredoxin 4 deficiency induces accelerated ovarian aging through destroyed proteostasis in granulosa cells.

Ovarian aging, a complex and challenging concern within the realm of reproductive medicine, is associated with reduced fertility, menopausal symptoms and long-term health risks. Our previous investigation revealed a correlation between Peroxiredoxin 4 (PRDX4) and human ovarian aging. The purpose of this research was to substantiate the protective role of PRDX4 against ovarian aging and elucidate the underlying molecular mechanism in mice. In this study, a Prdx4-/- mouse model was established and it was observed that the deficiency of PRDX4 led to only an accelerated decline of ovarian function in comparison to wild-type (WT) mice. The impaired ovarian function observed in this study can be attributed to an imbalance in protein homeostasis, an exacerbation of endoplasmic reticulum stress (ER stress), and ultimately an increase in apoptosis of granulosa cells. Furthermore, our research reveals a noteworthy decline in the expression of Follicle-stimulating hormone receptor (FSHR) in aging Prdx4-/- mice, especially the functional trimer, due to impaired disulfide bond formation. Contrarily, the overexpression of PRDX4 facilitated the maintenance of protein homeostasis, mitigated ER stress, and consequently elevated E2 levels in a simulated KGN cell aging model. Additionally, the overexpression of PRDX4 restored the expression of the correct spatial conformation of FSHR, the functional trimer. In summary, our research reveals the significant contribution of PRDX4 in delaying ovarian aging, presenting a novel and promising therapeutic target for ovarian aging from the perspective of endoplasmic reticulum protein homeostasis.

Peroxiredoxin-4 and Dopamine D5 Receptor Interact to Reduce Oxidative Stress and Inflammation in the Kidney.

Aims: Reactive oxygen species are highly reactive molecules generated in different subcellular compartments. Both the dopamine D5 receptor (D5R) and endoplasmic reticulum (ER)-resident peroxiredoxin-4 (PRDX4) play protective roles against oxidative stress. This study is aimed at investigating the interaction between PRDX4 and D5R in regulating oxidative stress in the kidney. Results: Fenoldopam (FEN), a D1R and D5R agonist, increased PRDX4 protein expression, mainly in non-lipid rafts, in D5R-HEK 293 cells. FEN increased the co-immunoprecipitation of D5R and PRDX4 and their colocalization, particularly in the ER. The efficiency of Förster resonance energy transfer was increased with FEN treatment measured with fluorescence lifetime imaging microscopy. Silencing of PRDX4 increased hydrogen peroxide production, impaired the inhibitory effect of FEN on hydrogen peroxide production, and increased the production of interleukin-1ß, tumor necrosis factor (TNF), and caspase-12 in renal cells. Furthermore, in Drd5-/- mice, which are in a state of oxidative stress, renal cortical PRDX4 was decreased whereas interleukin-1ß, TNF, and caspase-12 were increased, relative to their normotensive wild-type Drd5+/+ littermates. Innovation: Our findings demonstrate a novel relationship between D5R and PRDX4 and the consequent effects of this relationship in attenuating hydrogen peroxide production in the ER and the production of proinflammatory cytokines. This study provides the potential for the development of biomarkers and new therapeutics for renal inflammatory disorders, including hypertension. Conclusion: PRDX4 interacts with D5R to decrease oxidative stress and inflammation in renal cells that may have the potential for translational significance. Antioxid. Redox Signal. 38, 1150-1166.

Target proteins profiling of irreversible kinase inhibitor pelitinib and discovery of degradation of PRDX4 by label free chemoproteomics.

Cell-based methods for profiling the kinase inhibitor selectivity are badly needed, especially for the irreversible kinase inhibitors. Here we reported a chemoproteomics approach for profiling the target proteins of irreversible kinase inhibitor with label free quantitative proteomics by using iodoacetamide alkyne as a chemical probe. In total 41 proteins were identified in high confidence (fold change 3.5, p value < 0.05) including PRDX4, STAT3, E2 conjugating enzymes UBE2L3, UBE2K, UBE2N, UBE2V1 and UBE2Z as well as E3 ligase TRIM 25. We validated the interaction between pelitinib and PRDX4 with a cell-based assay, and discovered that pelitinib can induce the degradation of PRDX4. The discovery was confirmed by biochemical assay, cellular thermal shift assay and miRNA knockdown experiment. Our data suggested that pelitinib can be a covalent molecular glue inducing the degradation of PRDX4. In addition, our work demonstrated that identification of the interactions between ligand and ubiquitylation associated proteins by chemoproteomics profiling can be used as a new strategy for identifying molecular glue degraders.