Peroxiredoxin Research Articles

α-Pinene Improves Follicle Morphology and Increases the Expression of mRNA for Nuclear Factor Erythroid 2-Related Factor 2 and Peroxiredoxin 6 in Bovine Ovarian Tissues Cultured In Vitro.

Oxidative stress during in vitro of ovarian tissues has adverse effects on follicle survival. α-pinene is a monoterpenoid molecule with antioxidant activity that has great potential to maintain cell survival in vitro. This study investigated the effect of α-pinene (1.25, 2.5, 5.0, 10.0, or 20.0 μg/mL) on primordial follicle growth and morphology, as well as on stromal cells and collagen fibers in bovine ovarian slices cultured for six days. The effect of α-pinene on transcripts of catalase (CAT), superoxide dismutase (SOD), peroxiredoxin 6 (PRDX6), glutathione peroxidase (GPX1), and nuclear factor erythroid 2-related factor 2 (NRF2) was investigated by real-time PCR. The tissues were processed for histological analysis to evaluate follicular growth, morphology, stromal cell density, and collagen fibers. The results showed that 2.5, 5.0, or 10.0 µg/mL α-pinene increased the percentages of normal follicles but did not influence follicular growth. The α-pinene (10.0 µg/mL) kept the stromal cell density and collagen levels in cultured bovine ovarian tissue like uncultured tissues. Ovarian tissues cultured in control medium had reduced expression of mRNA for NRF2, SOD, CAT, GPX1, and PRDX6, but α-pinene (10.0 µg/mL) increased mRNA levels for NRF2 and PRDX6. In conclusion, 10.0 µg/mL α-pinene improves the follicular survival, preserves stromal cell density and collagen levels, and increases transcripts of NRF2 and PRDX6 after in vitro culture of bovine ovarian tissue.

Dynamics in Redox-Active Molecules Following Ischemic Preconditioning in the Brain.

It is well known that the brain is quite vulnerable to oxidative stress, initiating neuronal loss after ischemia-reperfusion (IR) injury. A potent protective mechanism is ischemic preconditioning (IPC), where proteins are among the primary targets. This study explores redox-active proteins' role in preserving energy supply. Adult rats were divided into the control, IR, and IPC groups. Protein profiling was conducted to identify modified proteins and then verified through activity assays, immunoblot, and immunohistochemical analyses. IPC protected cortex mitochondria, as evidenced by a 2.26-fold increase in superoxide dismutase (SOD) activity. Additionally, stable core subunits of respiratory chain complexes ensured sufficient energy production, supported by a 16.6% increase in ATP synthase activity. In hippocampal cells, IPC led to the downregulation of energy-related dehydrogenases, while a significantly higher level of peroxiredoxin 6 (PRX6) was observed. Notably, IPC significantly enhanced glutathione reductase activity to provide sufficient glutathione to maintain PRX6 function. Astrocytes may mobilize PRX6 to protect neurons during initial ischemic events, by decreased PRX6 positivity in astrocytes, accompanied by an increase in neurons following both IR injury and IPC. Maintained redox signaling via astrocyte-neuron communication triggers IPC's protective state. The partnership among PRX6, SOD, and glutathione reductase appears essential in safeguarding and stabilizing the hippocampus.

Metabolomics and microbiome co-analysis reveals altered innate immune responses in Charybdis japonica following Aeromonas hydrophila infection

A comprehensive bioinformatics analysis was conducted to elucidate the innate immune response of Charybdis japonica following exposure to Aeromonas hydrophila. This study integrated metabolomics, 16S rRNA sequencing, and enzymatic activity data to dissect the immune mechanisms activated in response to infection. Infection with A. hydrophila resulted in an increased abundance of beneficial intestinal genera such as Photobacterium spp., Rhodobacter spp., Polaribacter spp., Psychrilyobacter spp., and Mesoflavibacter spp. These probiotics appear to suppress A. hydrophila colonization by competitively dominating the intestinal microbiota. Key metabolic pathways affected included fatty acid biosynthesis, galactose metabolism, and nitrogen metabolism, highlighting their role in the crab's intestinal response. Enzymatic analysis revealed a decrease in activities of hexokinase, phosphofructokinase, and pyruvate kinase, which are essential for energy homeostasis and ATP production necessary for stress responses. Additionally, reductions were observed in the activities of acetyl-CoA carboxylase and fatty acid synthase. Gene expression analysis showed downregulation in Peroxiredoxin 1 (PRDX1), Peroxiredoxin 2 (PRDX2), glutathione-S-transferase (GST), catalase (CAT), and glutathione (GSH), with concurrent increases in malondialdehyde (MDA) levels, indicating severe oxidative stress. This study provides insights into the molecular strategies employed by marine crabs to counteract bacterial invasions in their natural habitat.

Mitochondrial Peroxiredoxin 6 Declines with Aging in Parallel with Increases in Hydrogen Peroxide Generation

Oxidative stress in cells and tissues arise from an imbalance between the generation of free radicals and the ability to detoxify these reactive oxygen species (ROS). Peroxiredoxins (PRDXs) are a family of widely distributed enzymes that act as master antioxidant regulators by scavenging peroxides and peroxynitrites. The accumulation of oxidative damage from chronic exposure to oxidative stress such as on DNA, mitochondrial proteins, and lipid macromolecules, is postulated to result in age-associated pathologies and physiological dysfunctions. Peroxiredoxin-6 (PRDX6) contributes to the maintenance of redox balance in a variety of tissues from lung to skeletal muscle. Here, we hypothesized that PRDX6 localizes to the mitochondria and plays a significant role in maintaining mitochondrial function through targeting mitochondrially generated ROS, and that gradual loss of mitochondrial PRDX6 contributes to increased oxidative stress and accumulation of oxidative damage during aging. In this study, respiratory rates and complex activities in isolated skeletal muscle mitochondria obtained from young (3-6 months) and old (20-24 months) C57/BL6J mice were compared. Amplex UltraRed fluorescence was used to determine production rates of mitochondrial hydrogen peroxide (H2O2). Western blot analysis was used to compare protein abundance and ELISA was used to measure lipid peroxidation in skeletal muscle mitochondrial fractions. Our findings revealed that isolated mitochondria from skeletal muscle in old mice generated higher rates of H2O2 from complex I (CI) during state 3 and state 4 (State 3; State 4) derived respiration (3.93 ± 0.41; 3.56 ± 0.38 pmol H2O2..s−1.mg−1) compared to young mouse mitochondria (1.57 ± 0.48; 1.89 ± 0.59 pmol H2O2.s−1.mg−1, p ≤ 0.05). This was also found when saturating amounts of ADP were used to stimulate maximal rates of oxygen consumption (old: 3.65 ± 0.77; young 1.40 ± 0.21 pmol H2O2−1.mg−1, p ≤ 0.05). Furthermore, CI activity was found to be lower in isolated mitochondria from old mice (0.65 ± 0.006 U.ug−1) compared to young mice (2.11 ± 0.64 U.ug−1, p ≤ 0.05). Similarly, mitochondrial PRDX6 abundance was lower while levels of lipid peroxidation product malondialdehyde were higher in skeletal muscle from old animals (old: 2712 ± 1320 pg.mL−1; young: 489 ± 225 pg.mL−1). These results show higher mitochondrial ROS generation rates and oxidative damage in old animals. These data also suggest that loss of PRDX6 in aging leads to mitochondrial dysfunction mediated by diminished CI activity and loss of membrane integrity. We are currently conducting mechanistic studies in cellular models with disrupted PRDX6 expression to confirm the effect of this protein in maintaining mitochondrial function. This study offers novel observations on the significance of mitochondrially localized PRDX6 in antioxidant defense as well as the sources and roles of ROS generation in contributing to an aging phenotype. National Institute of General Medical Sciences (NIGMS, Award Number R35GM146951). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peroxiredoxin 1 promotes proliferation and inhibits differentiation of MC3T3-E1 cells via AKT1 / NF-κB signaling pathway

ObjectivesOsteoporosis is the most common metabolic bone disease worldwide. The decrease in bone mass is primarily accompanied by a decrease in the number and activity of osteoblasts. Peroxiredoxins (PRDXs) are proteins that detect extremely low peroxide levels and act as sensors that regulate oxidation signals, thereby regulating various cellular functions. This study aimed to evaluate the effects of PRDX1 and estrogen on the biological behavior of osteoblasts, including their proliferation and differentiation. MethodsOvariectomized (OVX) mice were used to establish a model of osteoporosis and perform morphological and immunohistochemical analyses. Prdx1 gene knockout and overexpression were performed in mouse MC3T3-E1 pre-osteoblasts to assess proliferation and osteogenic differentiation using the cell counting kit-8, quantitative reverse transcription polymerase chain reaction, western blotting (WB), Alizarin Red S staining, etc. ResultsThe OVX mice exhibited osteoporosis and PRDX1 expression increased. In vitro experiments showed that during the osteogenic differentiation of osteoblasts, PRDX1 expression decreased, while the expression of COL1 and RUNX2 increased. After Prdx1 knockout, the proliferation of osteoblasts decreased; expression of Runx2, ALP, and COL1 increased; and mineralization increased. However, after Prdx1 overexpression, osteoblast proliferation was enhanced, whereas osteogenic differentiation and mineralization were inhibited. Estrogen inhibits the H2O2-induced decrease in osteoblastic differentiation and increase in PRDX1 expression. WB revealed that when LY294002 inhibited the AKT signaling pathway, the levels of p-AKT1, p-P65, and PRDX1 protein in MC3T3-E1 cells decreased. However, when pyrrolidine dithiocarbamate (PDTC) inhibited the NF-κB signaling pathway, the expression of p-AKT1 and PRDX1 did not change except for a significant reduction of p-P65 expression. Furthermore, PDTC reversed the decreased expression of RUNX2, ALP, and COL1 caused by PRDX1 overexpression. ConclusionsPRDX1 promotes the proliferation of osteoblasts and inhibits osteogenic differentiation. Estrogen regulated osteoblastic differentiation by affecting the expression of PRDX1 in osteoblasts, and the effect is related to the AKT1/NF-κB signaling pathway.

Calcified Cartilage-Guided Identification of Osteogenic Molecules and Geometries.

Calcified cartilage digested by chondroclasts provides an excellent scaffold to initiate bone formation. We analyzed bioactive proteins and microarchitecture of calcified cartilage either separately or in combination and evaluated biomimetic osteogenic culture conditions of surface-coated micropatterning. To do so, we prepared a crude extract from porcine femoral growth plates, which enhanced in vitro mineralization when coated on flat-bottom culture dishes, and identified four candidate proteins by fractionation and mass spectrometry. Murine homologues of two candidates, desmoglein 4 (DSG4) and peroxiredoxin 6 (PRDX6), significantly promoted osteogenic activity based on in vitro mineralization and osteoblast differentiation. Moreover, we observed DSG4 and PRDX6 protein expression in mouse femur. In addition, we designed circular, triangular, and honeycomb micropatterns with 30 or 50 μm units, either isolated or connected, to mimic hypertrophic chondrocyte-sized compartments. Isolated, larger honeycomb patterns particularly enhanced osteogenesis in vitro. Mineralization on micropatterns was positively correlated with the reduction of osteoblast migration distance in live cell imaging. Finally, we evaluated possible combinatorial effects of coat proteins and micropatterns and observed an additive effect of DSG4 or PRDX6 coating with micropatterns. These data suggest that combining a bioactive surface coating with osteogenic micropatterns may recapitulate initiation of bone formation during endochondral ossification.

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.

61 The first-in-class peroxiredoxin 3 (PRX3) inhibitor RSO-021 modulates the mesenchymal and immune phenotype in mesothelioma

61 The first-in-class peroxiredoxin 3 (PRX3) inhibitor RSO-021 modulates the mesenchymal and immune phenotype in mesothelioma

Role of Peroxiredoxin 1 Induced by Epstein-Barr Virus Infection in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC), a common cancer in Southern China, is associated with Epstein-Barr Virus (EBV) infection. Although many therapies for NPC have been established, the definite role of EBV in NPC remains unclear. Therefore, this work focuses on LMP2A, a latent EBV gene, and investigates whether LMP2A is related to peroxiredoxin 1 (PRDX1) in EBV-positive NPC. The mRNA and protein expression levels of LMP2A, PRDX1, and beta-catenin were compared in patient samples. To identify molecular mechanisms, EBV-negative NP69 and EBV-positive C666-1 NPC cell lines were used. After making an agar cell block for cell slides, the intensity of LMP2A expression was observed visually. To measure the level of reactive oxygen species, both fluorescence microscope and flow cytometry were used. To investigate the intracellular signaling molecular mechanisms with and without the LMP2A gene, reverse transcription polymerase chain reaction and western blotting were used. Both patient samples and cells of nasopharyngeal carcinoma infected with EBV had increased expression of LMP2A compared with controls, and high ROS levels were identified. Cell viability assay showed that LMP2A promoted cell growth by regulating gene expression. Furthermore, LMP2A induced the expression of PRDX1 and beta-catenin. LMP2A also increased the expression of both cyclin B1 and cyclin D1. In NPC cells, PRDX1 and beta-catenin were regulated through LMP2A expression, which reduced cell growth through cell cycle-related gene expression. This study suggests that LMP2A could be a target molecule for inhibiting cancer progression in NPC cells infected with EBV.

PRDX6 alleviated heart failure by inhibiting doxorubicin-induced ferroptosis through the JAK2/STAT1 pathway inactivation.

Peroxiredoxin 6 (PRDX6) is a protective biomarker associated with ferroptosis in heart failure (HF). This study investigated the specific mechanism of PRDX6 on doxorubicin (DOX)-induced ferroptosis in HF. Wistar rats and H9c2 cells were induced by DOX to construct HF models. Pathological changes and collagen deposition in myocardium were investigated using HE and Masson staining. PRDX6 levels, indexes of ferroptosis, and JAK2/STAT1 pathway were detected by qRT-PCR, Western blot, and biochemical kits. DOX promoted heart weight/body weight, increased inflammation and collagen deposition, increased PTGS2 and MDA levels, and decreased SLC7A11, GPX4, FTH1, and PRDX6 levels in myocardium. PRDX6 overexpression reduced PTGS2, MDA, Fe2+, and LDH levels, inhibited JAK2 and STAT1 phosphorylation, and increased SLC7A11, GPX4, and FTH1 levels in DOX-added H9c2 cells. RO8191 and erastin reversed the inhibition of PRDX6 on ferroptosis through the JAK2/STAT1 pathway. Overall, PRDX6 alleviated HF by inhibiting DOX-induced ferroptosis through the JAK2/STAT1 pathway inactivation.

Abstract 3269: APE1/NF-κB/PRDX2 activated by reflux conditions mediates chemoresistance in esophageal adenocarcinoma by suppressing ferroptosis

Abstract Background: Esophageal adenocarcinoma (EAC) stands out as one of the most lethal malignancies, characterized by a discouragingly low 5-year survival rate of less than 20%. Accumulating lines of evidence indicate that ferroptosis, a novel type of cell death, can regulate drug resistance. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, plays an antioxidant protective role in the cells under stress by regulating redox homeostasis. We aimed to elucidate the role of PRDX2 in regulating reactive oxygen species (ROS), ferroptosis, and chemoresistance in EAC. Methods: In this investigation, we analyzed RNA sequencing data and public databases to uncover the aberrant overexpression and potential chemoresistance function of PRDX2 in EAC. We employed transient acidic bile salts exposure (ABS) and repeated ABS exposure (rABS) to mimic gastroesophageal reflux disease, the main risk factor for EAC, in in vitro cell models. EAC cells and 3D organotypic culture (OTC) served as platforms to delve into the anti-ferroptosis function and regulatory mechanisms of PRDX2. The findings were validated using patient-derived xenografts (PDX) and human EAC tissue microarrays (TMA). Results: Elevated PRDX2 level was observed in both EAC patients and cell lines exhibiting poor responsiveness to chemotherapy. Intrinsic overexpression of PRDX2 emerged as a crucial factor in the recovery from ABS-exposure-induced lipid peroxidation. Silencing PRDX2 heightened the sensitivity of EAC cells to Oxaliplatin by inhibiting ferroptosis in a GPX4-dependent manner. Mechanistically, we found ABS activate the APE1/redox/NF-kB signal axis, leading to the upregulation of PRDX2 transcription. The co-overexpression of APE1 and PRDX2 was identified through both immunofluorescent staining in cells/OTC and immunohistochemistry staining in TMA. Moreover, the APE1-redox-specific inhibitor, APX2009, significantly sensitized EAC cells to Oxaliplatin by repressing PRDX2 and inducing ferroptosis. Remarkably, the combination therapy of Oxaliplatin and APX2009 achieved a synergistic effect in EAC-PDX tumors. Conclusion: The study unveils a novel signaling axis, APE1/NF-kB/PRDX2, linking disease-associated ROS unbalance and ferroptosis-related chemoresistance. Targeting PRDX2 signaling by APE1-redox-specific inhibition provides a potential novel strategy for combined chemotherapies in refractory EAC. Citation Format: Lei Chen, Heng Lu, Farah Ballout, Dunfa Peng, Zheng Chen, Jianwen Que, Steven Chen, Oliver McDonald, Alexander Zaika, Wael El-Rifai. APE1/NF-κB/PRDX2 activated by reflux conditions mediates chemoresistance in esophageal adenocarcinoma by suppressing ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3269.

Abstract 384: SLC7A11 modulates sensitivity to the first-in-class mitochondrial peroxiredoxin 3 inhibitor thiostrepton (RSO-021) via a ferroptosis independent pathway

Abstract Mitochondrial peroxiredoxin 3 (PRX3) has been identified as an actionable cancer vulnerability that is currently being investigated in the first-in-human phase 1 clinical trial, MITOPE (NCT05278975). RSO-021 (thiostrepton or TS) is a redox-active drug that inhibits PRX3’s peroxidase activity via covalent adduction of its active site peroxidatic and resolving cysteine residues, forming an irreversible crosslink across the protein dimer. Covalent inhibition of PRX3 results in tumor cell death due to a diminished ability to remove high levels of mitochondrial reactive oxygen species (ROS) in cancer cells. To better understand the mechanism underpinning sensitivity and resistance, TS-tolerant human mesothelioma cell lines were generated under constant TS pressure and subjected to a drug-screen comprising chemotherapeutic agents, metabolic inhibitors, ROS modulating agents and ferroptosis-inducing compounds, to identify synergistic interactions compared with TS- sensitive cell lines. Erastin, an SLC7A11 inhibitor exhibited the greatest synergy when combined with TS, potentiated PRX3 covalent crosslinking and cellular ROS levels. Synergy with TS was enhanced by cystine depletion and could be phenocopied by siRNA knockdown of SLC7A11. SLC7A11 was up-regulated in TS-tolerant cells and resistance could be overcome with erastin. TS-tolerant cell lines have significantly reduced glutathione levels and slowed proliferation. Ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 did not inhibit TS or TS in combination with erastin. In summary, SLC7A11 up-regulation confers tolerance to TS through a mechanism involving a cysteine-dependent modulation of the redox state independent of canonical ferroptosis, that can be overcome by the addition of an SLC7A11 inhibitor. The correlation between SLC7A11 with response to TS in a 33-patient primary mesothelioma explant co-clinical trial, and patients enrolled into the MITOPE phase 1/2 trial will be presented. Citation Format: Terri Messier, Victoria Gibson, Aleksandra Bzura, Joanna Dzialo, Charlotte Poile, Stephanie Stead, Alexis Saaman, George N. Naumov, Dean A. Fennell, Brian Cunniff. SLC7A11 modulates sensitivity to the first-in-class mitochondrial peroxiredoxin 3 inhibitor thiostrepton (RSO-021) via a ferroptosis independent pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 384.

Abstract 4716: First-in-class peroxiredoxin 3 (PRX3) inhibitor RSO-021 triggers mesenchymal-to-epithelial transition in mesothelioma

Abstract Mesothelioma, a lethal cancer associated with asbestos exposure, lacks effective drug therapy particularly in the relapsed treatment setting. Epithelial-to-mesenchymal transition (EMT) confers an aggressive sarcomatoid phenotype capable of invasion, metastasis, and drug resistance. Pharmacological targeting of EMT could favorably alter the progression and treatment of mesothelioma. We identified a geospatial transcriptomic gradient between adjacent epithelioid and sarcomatoid regions in patient-derived biphasic mesotheliomas involving hypoxia tolerance, TGF-β, mitochondrial oxidative phosphorylation, and EMT. Mesothelioma cells rely on buffering of mitochondrial oxidative stress through the expression and activity of mitochondrial peroxiredoxin 3 (PRX3), which is covalently inactivated by the first-in-class inhibitor RSO-021, now under investigation in the phase 1/2 MITOPE clinical trial (NCT05278975). To better understand the pharmacodynamics of RSO-021, transcriptomic profiles of drug sensitive and resistant biphasic mesothelioma cell lines were generated. Differential analysis revealed that RSO-021 induced up-regulation of ROS response and apoptosis signaling, while down-regulating TGF-β and EMT markers, WNT signaling, and TEAD target genes. Additionally, we observed altered expression of the mesothelioma specific EMT genes Col5A2, mesothelin and VISTA. Resistance to RSO-021 conferred significant proliferative defects and was reversible upon RSO-021 removal, however the down-regulation of EMT genes was conserved, suggesting an irreversible mesenchymal-to-epithelial transition. In summary PRX3 is a clinically actionable drug target, inhibition of which correlates with EMT modulation. This activity suggests a mechanism to alter mesothelioma progression and prevent the emergence of the most aggressive mesothelioma phenotype. Citation Format: Terri Messier, Victoria Gibson, David J. Seward, Essa Y. Baitei, Peter Wells Jordan, Charlotte Poile, Joanna Dzialo, Aleksandra Bzura, Kudzayi Kutywayo, Apostolos Nakas, George N. Naumov, Dean A. Fennell, Brian Cunniff. First-in-class peroxiredoxin 3 (PRX3) inhibitor RSO-021 triggers mesenchymal-to-epithelial transition in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4716.

Chemoproteomic Profiling Maps Zinc-Dependent Cysteine Reactivity.

As a vital micronutrient, zinc is integral to the structure, function, and signaling networks of diverse proteins. Dysregulated zinc levels, due to either excess intake or deficiency, are associated with a spectrum of health disorders. In this context, understanding zinc-regulated biological processes at the molecular level holds significant relevance to public health and clinical practice. Identifying and characterizing zinc-regulated proteins in their diverse proteoforms, however, remain a difficult task in advancing zinc biology. Herein, we address this challenge by developing a quantitative chemical proteomics platform that globally profiles the reactivities of proteinaceous cysteines upon cellular zinc depletion. Exploiting a protein-conjugated resin for the selective removal of Zn2+ from culture media, we identify an array of zinc-sensitive cysteines on proteins with diverse functions based on their increased reactivity upon zinc depletion. Notably, we find that zinc regulates the enzymatic activities, post-translational modifications, and subcellular distributions of selected target proteins such as peroxiredoxin 6 (PRDX6), platelet-activating factor acetylhydrolase IB subunit alpha1 (PAFAH1B3), and phosphoglycerate kinase (PGK1).

A novel marine-derived anti-acute kidney injury agent targeting peroxiredoxin 1 and its nanodelivery strategy based on ADME optimization

Insufficient therapeutic strategies for acute kidney injury (AKI) necessitate precision therapy targeting its pathogenesis. This study reveals the new mechanism of the marine-derived anti-AKI agent, piericidin glycoside S14, targeting peroxiredoxin 1 (PRDX1). By binding to Cys83 of PRDX1 and augmenting its peroxidase activity, S14 alleviates kidney injury efficiently in Prdx1-overexpression (Prdx1-OE) mice. Besides, S14 also increases PRDX1 nuclear translocation and directly activates the Nrf2/HO-1/NQO1 pathway to inhibit ROS production. Due to the limited druggability of S14 with low bioavailability (2.6%) and poor renal distribution, a pH-sensitive kidney-targeting dodecanamine-chitosan nanoparticle system is constructed to load S14 for precise treatment of AKI. l-Serine conjugation to chitosan imparts specificity to kidney injury molecule-1 (Kim-1)-overexpressed cells. The developed S14-nanodrug exhibits higher therapeutic efficiency by improving the in vivo behavior of S14 significantly. By encapsulation with micelles, the AUC0‒t, half-life time, and renal distribution of S14 increase 2.5-, 1.8-, and 3.1-fold, respectively. The main factors contributing to the improved druggability of S14 nanodrugs include the lower metabolic elimination rate and UDP-glycosyltransferase (UGT)-mediated biotransformation. In summary, this study identifies a new therapeutic target for the marine-derived anti-AKI agent while enhancing its ADME properties and druggability through nanotechnology, thereby driving advancements in marine drug development for AKI.

Expression and clinical role of PRDX6 in lung adenocarcinoma.

To explore the levels of expression and clinical role of peroxiredoxin 6 (PRDX6) in lung adenocarcinoma. 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. 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. PRDX6 is highly expressed in lung adenocarcinoma and higher levels of expression of the PRDX6 gene were associated with a poorer prognosis.

Melatonin improves the viability and ultrastructure of bovine oocyte-granulosa complexes of invitro cultured early antral follicles.

This study aims to investigate the effects of melatonin on follicular growth, viability and ultrastructure, as well as on the levels of mRNA for antioxidant enzymes, reactive oxygen species (ROS) and meiotic progression in oocytes from invitro cultured bovine early antral follicles. To this end, isolated early antral follicles (500-600 μm) were cultured in TCM-199+ alone or supplemented with 10-6 , 10-7 or 10-8 M melatonin at 38.5°C with 5% CO2 for 8 days. Follicle diameters were evaluated at days 0, 4 and 8 of culture. At the end of culture, ultrastructure, chromatin configuration, viability (calcein-AM and ethidium homodimer-1 staining), and the levels of ROS and mRNA for catalase (CAT), superoxide dismutase (SOD) and peroxiredoxin 6 (PRDX6) and glutathione peroxidase (GPx) were investigated in oocyte-granulosa cell complexes (OGCs). The results showed that early antral follicles cultured with 10-6 and 10-8 M melatonin had a progressive and significant increase in their diameters throughout the culture period (p < .05). Additionally, oocytes from follicles cultured with 10-7 or 10-8 M melatonin had increased fluorescence for calcein-AM, while those cultured with 10-6 or 10-7 M had reduced fluorescence for ethidium homodimer-1. Different from follicles cultured in other treatments, those cultured with 10-8 M melatonin had well-preserved ultrastructure of oocyte and granulosa cells. Melatonin, however, did not influence the levels of ROS, the mitochondrial activity, oocyte meiotic resumption and expression mRNA for SOD, CAT, GPX1 and PRDX6. In conclusion, the presence of 10-8 M melatonin in culture medium improves viability and preserves the ultrastructure of oocyte and granulosa cells of early antral follicles cultured invitro.

Concomitant inhibition of the thioredoxin system and nonhomologous DNA repair potently sensitizes Philadelphia-positive lymphoid leukemia to tyrosine kinase inhibitors.

Breakpoint cluster region-Abelson (BCR::ABL1) gene fusion is an essential oncogene in both chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). While tyrosine kinase inhibitors (TKIs) are effective in up to 95% of CML patients, 50% of Ph+ B-ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B-ALL. Previous studies have shown that inhibitors of the thioredoxin (TXN) system exert antileukemic activity against B-ALL cells, particularly in combination with other drugs. Here, we present that peroxiredoxin-1 (PRDX1), one of the enzymes of the TXN system, is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. PRDX1 knockout negatively affects the viability of Ph+ B-ALL cells and sensitizes them to TKIs. Analysis of global gene expression changes in imatinib-treated, PRDX1-deficient cells revealed that the nonhomologous end-joining (NHEJ) DNA repair is a novel vulnerability of Ph+ B-ALL cells. Accordingly, PRDX1-deficient Ph+ B-ALL cells were susceptible to NHEJ inhibitors. Finally, we demonstrated the potent efficacy of a novel combination of TKIs, TXN inhibitors, and NHEJ inhibitors against Ph+ B-ALL cell lines and primary cells, which can be further investigated as a potential therapeutic approach for the treatment of Ph+ B-ALL.

Exploring genetic polymorphisms and transcript levels of antioxidant and metabolic markers for prediction and monitoring diarrhea in Holstein dairy calves

ABSTRACT The objective of this study was to investigate the genetic polymorphisms and transcript levels of antioxidant and metabolic markers for diarrhea monitoring in Holstein dairy calves. For DNA and RNA extraction, blood samples were taken from 100 female calves (50 with diarrhea and 50 appearing healthy). DNA sequencing and real time PCR for and metabolic genes were performed to identify genetic variants linked to diarrhea susceptibility. Results revealed that there were nucleotide sequence variations in the investigated genes between healthy and diarrheal calves. Keap1, HMOX1, CMPK2, ASPG, FPGT, TNNI3 K, and LPCAT1 were considerably more highly expressed in diarrheic calves than resistant ones. The Nrf2, PRDX2, and PRDX6 genes, however, produced a different pattern. This study provides a practical control strategy to lessen newborn diarrhea in Holstein dairy calves and emphasizes the significance of nucleotide variations along with antioxidant and metabolic gene expression patterns as proxy markers of diarrhea susceptibility/resistance.

Huashi Baidu formula alleviates lipopolysaccharide-induced inflammation and acute lung injury in mice by targeting nuclear factor κB/phosphatidylinositol 3-kinase and peroxiredoxin 5

Abstract Background Acute respiratory distress syndrome induced by acute lung injury (ALI) is the main cause for the high mortality of coronavirus disease 2019 (COVID-19). Huashi Baidu formula (HSBD) with the effects of eliminating dampness, clearing heat, ventilating lung, and removing toxin has been proven to be effective in the treatment of COVID-19, especially in severe cases. However, the underlying mechanism and target proteins of HSBD remain unclear. Objective To provide evidence and decipher the mechanism of HSBD in alleviating inflammation and ALI. Materials and Methods A mouse model of ALI was induced by lipopolysaccharide (LPS), and hematoxylin-eosin staining was used to examine the protective effects of HSBD on the model mice. The cellular thermal shift assay and proteomics analysis were used to predict the target proteins. Furthermore, the A549 cells with peroxiredoxin 5 (PRDX5) knockdown were established to validate the predicted proteins. Results Huashi Baidu formula treatment mitigated ALI and inflammatory cytokine dysfunction in LPS-induced mice, thus exerting a therapeutic effect on COVID-19. Huashi Baidu formula could serve as a therapeutic agent to alleviate inflammation and lung injury via nuclear factor κB and phosphatidylinositol 3-kinase signaling and interleukin 17 inhibition as well as targeting PRDX5, which could be one of the promising targets for treating inflammation. In the A549 cell line with PRDX5 knockdown (si-Prdx5), the anti-inflammation effects of HSBD, including reversing LPS-induced increase in the nitric oxide level and reduction in the hydrogen peroxide content, were attenuated. Thus, HSBD protected A549 cells from LPS-induced inflammation mainly by targeting PRDX5. Conclusions Huashi Baidu formula alleviates ALI by targeting nuclear factor κB/phosphatidylinositol 3-kinase and PRDX5, as well as inhibiting the immune response induced by IL-17.