Thiol-specific Antioxidant Research Articles

Molecular characterization, cytoprotective, DNA protective, and immunological assessment of peroxiredoxin-1 (Prdx1) from yellowtail clownfish (Amphiprion clarkii)

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.

Review of Postmortem Protein Oxidation in Skeletal Muscle and the Role of the Peroxiredoxin Family of Endogenous Antioxidants

The development of fresh meat quality is dictated by biochemical changes during the perimortem and postmortem period. Lipid and protein oxidation in postmortem skeletal muscle and meat products is detrimental to product quality. The mechanisms that influence lipid and protein oxidation in fresh meat remain unelucidated. Peroxiredoxins are thiol-specific antioxidant proteins that are highly reactive and abundant and may be involved in limiting oxidation early postmortem. This review aims to provide a background on oxidation in skeletal muscle, peroxiredoxins, a summary of proteomic experiments associating peroxiredoxins and meat quality, and the importance of context from proteomic methods and results. Additional controlled experiments considering the cellular conditions of postmortem skeletal muscle are necessary to further understand the contribution of peroxiredoxins to fresh meat quality development.

METABOLOMICS ANALYSIS IDENTIFIED AN ORALLY ACTIVE METABOLITE FOR TREATING CROHN’S DISEASE-ASSOCIATED INTESTINAL FIBROSIS

Abstract BACKGROUND Intestinal fibrosis is a debilitating complication of Crohn’s disease (CD) patients. Surgical resection is used to treat intestinal strictures, but this approach may adversely affect the patients' quality of life. Based on the findings of our groups and others, we hypothesize that intestinal metabolites are associated with intestinal fibrosis. We aimed to discover novel anti-fibrogenic metabolites. METHODS We compared the intestinal metabolite profiles between non-IBD, stricturing CD, and non-stricturing CD patients in an inflammatory bowel disease (IBD) multi-omics database to identify CD stricture-related metabolites. We compared the fecal metabolite profiles between trinitrobenzene sulfonic acid (TNBS)-treated mice with and without anti-fibrogenic CSA13 treatment to identify mouse intestinal fibrosis-related metabolites. RESULTS Compared to non-stricturing CD patients and non-IBD patients, stricturing CD patients had reduced fecal levels of inosine. Compared to normal mice, TNBS-treated mice had reduced fecal levels of inosine. CSA13-exposed TNBS-treated mice had increased fecal inosine levels. Oral administration of inosine (10 mg/kg/day for 14 days) effectively reduced ileal fibrosis in fibrotic 40-week-old SAMP1/YitFc mice with 96-100% reduction of intestinal collagen mRNA expression and 87% reduction of overall ileal disease activity. Although inosine did not directly inhibit collagen expression in primary human ileal fibroblasts from stricturing CD patients (CD-HIF), it reduced collagen mRNA expression in fresh ileal tissues from stricturing CD patients. RNA-seq and proteomics identified that peroxiredoxin 2 (PRDX2) is negatively associated with intestinal strictures in CD patients. PRDX2 is a thiol-specific antioxidant gene that reduces peroxides to alcohols. Inosine increased the secretion of peroxiredoxin 2 (PRDX2) in primary human intestinal epithelial cells (HPEC). The inosine-mediated PRDX2 secretion was inhibited by the pretreatment of adenosine 2A receptor (A2AR) inhibitor ZM241385. PRDX2 reduced cell stress, induced cell stress-related protein (cyclooxygenase 2/COX-2), and inhibited collagen expression in the CD-HIF. Inhibition of COX2 by indomethacin reversed the anti-fibrogenic effect of PRDX2 in the CD-HIF. Inhibition of PRDX2 caused increased collagen expression in the CD-HIF. Lentiviral overexpression of Prdx2 ameliorated ileal fibrosis in the fibrotic SAMP1/YitFc mice. Fibrotic SAMP1/YitFc mice had reduced circulating free thiol, reflecting increased systemic oxidative stress. Inosine restored circulating free thiol levels in SAMP1/YitFc mice. CONCLUSIONS Inosine is an orally active anti-fibrogenic metabolite that promotes PRDX2 expression from intestinal epithelial cells. PRDX2 is a newly identified anti-fibrogenic protein, which may inhibit collagen expression by reducing cell stress.

Molecular characterization and functional analysis of peroxiredoxin 4 in grass carp (Ctenopharyngodon idella)

Peroxiredoxins (Prxs) are a group of evolutionarily conserved selenium-independent thiol-specific antioxidant proteins. In this study, the peroxiredoxin-4 (CiPrx4) gene from grass carp was identified and characterized. The full-length of CiPrx4 is 1339 bp, encoding 260 amino acids that contain two peroxiredoxin signature motifs and two GVL motifs. CiPrx4 belongs to the typical 2-Cys subfamily and shows the highest homology with Prx4 from Cyprinus carpio (95.4%). CiPrx4 mRNA was constitutively expressed in all tested tissues and was upregulated by grass carp reovirus and pathogen-associated molecular pattern (PAMP) stimulation. CiPrx4 was localized in the cytoplasm and co-localized with the endoplasmic reticulum. The purified CiPrx4 protein protected DNA from degradation in a dose-dependent manner. Moreover, the overexpression of CiPrx4 in Escherichia coli and fish cells showed apparent antioxidant and antiviral activities. Collectively, the results of the present study provide new insights for further understanding the functions of Prx4 in teleost fish.

A comparative proteomic analysis of Candida species in response to the oxidizing agent cumene hydroperoxide

Candida genus comprises several species that can be found in the oral cavity and the gastrointestinal and genitourinary tracts of healthy individuals. Under certain conditions, however, they behave as opportunistic pathogens that colonize these tissues, most frequently when the immune system is compromised by a disease or under certain medical treatments. To colonize the human host, these organisms require to express cell wall proteins (CWP) that allowed them to adhere and adapt to the reactive oxygen (ROS) and nitrogen (RNS) species produced in the macrophage during the respiratory burst. The aim of this study was to determine how four Candida species respond to the oxidative stress imposed by cumene hydroperoxide (CHP). To this purpose, C. albicans, C. glabrata, C. krusei and C. parapsilosis were exposed to this oxidant which is known to generate ROS in the membrane phospholipids. Accordingly, both mock and CHP-exposed cells were used to extract and analyze CWP and also to measure catalase activity and the levels of protein carbonylation. Results indicated that all four species express different CWP to neutralize ROS. Most relevant among these proteins were the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase, known as moonlight proteins because in addition to participate in glycolysis they play an important role in the cell response to ROS. In addition, a thiol-specific antioxidant enzyme (Tsa) was also found to counteract ROS.

Transcriptional modifications and the cytoprotective, DNA protective, and wound healing effects of peroxiredoxin-1 from Sebastes schlegelii

Peroxiredoxins are a group of thiol-specific antioxidant proteins that take six isoforms in vertebrates and allow the innate immune system to sense and detoxify reactive oxygen species. In this study, we identified and characterized the perxiredoxin-1 (SsPrdx1) cDNA sequence from the rockfish, Sebastes schlegelii. In silico analysis revealed that SsPrdx1 contained a 594 bp long open reading frame (ORF) encoding a protein of 198 amino acids, with a predicted molecular weight and theoretical isoelectric point of 21.97 kDa and 6.30, respectively. The SsPrdx1 gene comprised six exons linked by five introns, while peroxiredoxin signature motifs were found in the highly conserved third, fourth, and fifth exons. Phylogenetic analysis and sequence alignment suggested that SsPrdx1 is evolutionarily conserved and that its most closely related counterpart is Salarias fasciatus. Recombinant SsPrdx1 (rSsPrdx1) displayed supercoiled DNA protection and insulin disulfide reduction activities in a concentration-dependent manner, while cells transiently transfected with pcDNA3.1 (+)/SsPrdx1 exhibited significant cytoprotective effects under oxidative stress and wound healing activity. SsPrdx1 transcripts were constitutively expressed under normal physiological conditions, with the highest expression observed in the blood. Moreover, SsPrdx1 expression increased in the blood, spleen, and liver following immune provocation by LPS, poly I:C, and Streptococcus iniae injection. Thus, this study provides insights into the role of SsPrdx1 in rockfish immune protection.

Analyses of the expression, immunohistochemical properties and serodiagnostic potential of Schistosoma japonicum peroxiredoxin-4

BackgroundSchistosoma japonicum, which inhabits the mesenteric vein of the mammalian hosts for about 20 to 30 years, is subjected to the oxidative stresses from the host defense mechanism during their intra-mammalian stages. To counteract this host immune attack, the parasite utilizes their antioxidant system for survival inside the host. Peroxiredoxins (Prxs), thiol-specific antioxidant proteins, play an essential role for protecting the parasite against oxidative stress by reducing hydrogen peroxide to water. Only three types of 2-Cys Prxs have been previously characterized in S. japonicum whereas a fourth Prx has been identified for Schistosoma mansoni as Prx-4. A sequence coding homologous to this gene in the S. japonicum database was identified, characterized and expressed as recombinant SjPrx-4 protein (rSjPrx-4). Furthermore, rSjPrx-4 was evaluated in this study for its diagnostic potentials in detecting S. japonicum infection in humans.ResultsThe gene found in the parasite genome contained 2 active-site cysteines with conserved sequences in the predicted amino acid (AA) sequence and showed 75% identity with that of the previously characterized Prx (TPx-1) of S. japonicum. The gene was expressed in different stages of schistosome life-cycle with highest transcription level in the adult male. The gene was cloned into a plasmid vector and then transfected into Escherichia coli for expression of rSjPrx-4. Anti-rSjPrx-4 mouse sera recognized native SjPrx-4 in egg and adult worm lysate by western blotting. The result of a mixed function oxidation assay in which rSjPrx-4 prevented the nicking of DNA from hydroxyl radicals confirmed its antioxidant activity. Subsequently, immunolocalization analysis showed the localization of SjPrx-4 inside the egg, on the tegument and in the parenchyma of the adult worm. Enzyme-linked immunosorbent assay results showed that rSjPrx-4 has 83.3% sensitivity and 87.8% specificity. Its diagnostic potential was further evaluated in combination with recombinant SjTPx-1 protein, yielding an improved sensitivity and specificity of 90% and 92.7%, respectively.ConclusionsThese results suggest that SjPrx-4 plays a role as an antioxidant dealing with oxidative stresses of S. japonicum, and its diagnostic potential improved by coupling it with SjTPx-1 is a proof for developing a serological test with better diagnostic performance for human schistosomiasis.

RNA-sequencing analysis revealed genes associated drought stress responses of different durations in hexaploid sweet potato

Purple-fleshed sweet potato (PFSP) is an important food crop, as it is a rich source of nutrients and anthocyanin pigments. Drought has become a major threat to sustainable sweetpotato production, resulting in huge yield losses. Therefore, the present study was conducted to identify drought stress-responsive genes using next-generation (NGS) and third-generation sequencing (TGS) techniques. Five cDNA libraries were constructed from seedling leaf segments treated with a 30% solution of polyethylene glycol (PEG-6000) for 0, 1, 6, 12, and 48 h for second-generation sequencing. Leaf samples taken from upper third of sweet potato seedlings after 1, 6, 12, and 48 h of drought stress were used for the construction of cDNA libraries for third-generation sequencing; however, leaf samples from untreated plants were collected as controls. A total of 184,259,679 clean reads were obtained using second and third-generation sequencing and then assembled into 17,508 unigenes with an average length of 1,783 base pairs. Out of 17,508 unigenes, 642 (3.6%) unigenes failed to hit any homologs in any databases, which might be considered novel genes. A total of 2, 920, 1578, and 2,418 up-regulated unigenes and 3,834, 2,131, and 3,337 down-regulated unigenes from 1 h, 6 h, 12 h, and 48 h library were identified, respectively in drought stress versus control. In addition, after 6, 12, and 48 h of drought stress, 540 up-regulated unigenes, 486 down-regulated unigenes and 414 significantly differentially expressed unigenes were detected. It was found that several gene families including Basic Helix-loop-helix (bHLH), basic leucine zipper (bZIP), Cystein2/Histidine2 (C2H2), C3H, Ethylene-responsive transcription factor (ERF), Homo domain-leucine zipper (HD-ZIP), MYB, NAC (NAM, ATAF1/2, and CUC2), Thiol specific antioxidant and WRKY showed responses to drought stress. In total, 17,472 simple sequence repeats and 510,617 single nucleotide polymorphisms were identified based on transcriptome sequencing of the PFSP. About 96.55% of the obtained sequences are not available online in sweet potato genomics resources. Therefore, it will enrich annotated sweet potato gene sequences and enhance understanding of the mechanisms of drought tolerance through genetic manipulation. Moreover, it represents a sequence resource for genetic and genomic studies of sweet potato.

Crystal structure of Akkermansiamuciniphila peroxiredoxin reveals a novel regulatory mechanism of typical 2-Cys Prxs by a distinct loop.

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins commonly found in organisms that protect cells from the damage of reactive oxygen species produced by metabolism and that participate in cell signaling. The Prx from the bacterium Akkermansiamuciniphila (AmPrx) is a typical 2-Cys Prx characterized by two conserved cysteines: Cys49 and Cys183. Here, we verified the peroxidase activity of AmPrx and determined its crystal structure in reduced form, which is a doughnut-shaped decamer composed of five dimers. Particularly, a distinct loop between the α4 helix and β6 strand is involved in the decameric interaction. Deletion of this loop destroys the decameric structure and significantly decreases the peroxidase activity of AmPrx. Our findings reveal a novel regulatory mechanism of typical 2-Cys Prx, in which the α4-β6 loop affects the assembly of Prx and, therefore, regulates its peroxidase activity.

Immunization against Leishmania major infection in BALB/c mice using a subunit-based DNA vaccine derived from TSA, LmSTI1, KMP11, and LACK predominant antigens.

Objective(s):To design a multivalent DNA vaccine encoding the most immunogenic regions of the Leishmania major antigens including TSA (Thiol-specific antioxidant protein), LmSTI1 (Leishmania major stress-inducible protein1), LACK (Leishmania homologue of receptors for activated C Kinase), and KMP11 (kinetoplastid membrane protein-11) on BALB/c mice.Materials and Methods:The chimeric construct was generated including the most immunogenic epitopes containing a combination of domains and oligopeptides of the aforementioned genes. The construct was cloned into pcDNA 3.1 plasmid and named “pleish-dom.” Following intramuscular injection of mice, the capability of the vector pleish-dom alone and with pIL-12 (expressing murine IL-12) to raise protective cytokines and parasite burden was evaluated in the BALB/c mice as a susceptible animal model against L. major.Results:The immunized mice with pleish-dom/pIL-12 showed the highest and the lowest levels of interferon-gamma (IFN-γ) and interleukin-10 (IL-10), as well as the lowest leishmanin skin test (LST) reactions, were found through 8 weeks post-infection.Conclusion:Although the obtained DNA vaccine from the immunogenic chimeric protein of L. major antigens was able to induce a high level of IFN-γ, it partially protected mice against L. major. However co-administration with pIL-12 led to shift immune response to Th1 phenotype, granuloma formation, and lowered parasite burden, and consequently distinct protection was found.

Cold-modulated small proteins abundance in winter triticale (x Triticosecale, Wittm.) seedlings tolerant to the pink snow mould (Microdochium nivale, Samuels and Hallett) infection.

Two winter triticale (x Triticosecale Wittmack) model cultivars: Hewo (tolerant to pink snow mould) and Magnat (sensitive) were used to test the effect of cold-hardening (4 weeks at 4°C) on soluble ≤50 kDa protein profiles of the seedling leaves. The presence and abundance of individual proteins were analysed via two-dimensional gel electrophoresis (2-DE) and Surface-Enhanced Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). Up to now, no proteomics analysis of triticale response to hardening has been performed. Thus, the present paper is the first in the series describing the obtained results. In our experiments, the exposure to the low temperature-induced only quantitative changes in the leaves of both cultivars, causing either an increase or decrease of 4-50 kDa protein abundance. Among proteins which were cold-accumulated in cv. Hewo's leaves, we identified two thioredoxin peroxidases (chloroplastic thiol-specific antioxidant proteins) as well as mitochondrial- β-ATP synthase subunit and ADP-binding resistance protein. On the contrary, in hardened seedlings of this genotype, we observed the decreased level of chloroplastic RuBisCO small subunit PW9 and epidermal peroxidase 10. Simultaneous SELDI-TOF analysis revealed several low mass proteins better represented in cold-hardened plants of tolerant genotype in comparison to the sensitive one and the impact of both genotype and temperature on their level. Based on those results, we suggest that indicated proteins might be potential candidates for molecular markers of cold-induced snow mould resistance of winter triticale and their role is worth to be investigated in the further inoculation experiments.

Abstract 1872: Oleuropein reduces Prdx1 expression, cell proliferation and viability in K562 human leukemia cells

Abstract Oleuropein is one of the two main phenolic compounds derived from olive leaves. Numerous studies have found that the polyphenols contained in olive leaves possess strong antioxidant properties, and increase apoptosis in many types of cancer cells, suggesting its possible efficacy as an anti-cancer treatments. The purpose of our study was to examine the effects of oleuropein extract on cell growth and viability of the K562 human leukemia cell line, and to investigate possible molecular mechanisms of action. Oleuropein of 98% purity was used for our studies. We first demonstrated that K562 cells treated with 200 and 400 μg/ml oleuropein exhibited a significant reduction in cell growth and viability after four days as compared to ethanol-treated controls. Cells treated with 200μg/ml oleuropein showed decreased cell proliferation and viability after two days, and exhibited approximately 90% reduction in cell density and viability after four days of treatment. A concentration of 400 μg/ml oleuropein resulted in 100% toxicity by day four. We also examined the effects of oleuropein on cellular cytotoxicity using an LDH release assay and found a two-fold elevation in LDH release within 24 hours of treatment with 200 μg/mL oleuropein. To begin to explore a mechanisms of action, we used western blotting to examine known antioxidant and signaling pathways for possible regulation by oleuropein in these cells. We examined expression of the Peroxiredoxin (Prdx) family of proteins, a group of thiol-specific antioxidants found to be elevated in many types of cancer. We found that 200 ug/ml oleuropein reduced Prdx1 expression by about 50% after eight and twenty-four hours, while having no significant effect on expression levels of Prdx-2, -3, -4, and -6. We are currently investigating the mechanism of cell death induced by oleuropein, as well as the role of Prdx1 in oleuropein-induced toxicity. Together with previously reported cancer-cell specific effects, this research provides a promising new avenue for the use of natural products as anti-cancer agents. Citation Format: Natalie Fulco, Sophia Fagan, Shelley A. Phelan. Oleuropein reduces Prdx1 expression, cell proliferation and viability in K562 human leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1872.

Cloning and molecular characterization of thiol-specific antioxidant gene of Leishmania tropica Turkey isolate

Background/aimThiol-specific antioxidant (TSA) protein is one of the most promising molecules among candidates for vaccine against cutaneous leishmaniasis. It was found to be significantly protective against different Leishmania species. In this study, cloning and molecular characterization of thiol-specific antioxidant gene of L. tropica Turkey isolate (LtTSA) were aimed. Materials and methods LtTSA was amplified by PCR using the specific primers of TSA gene and cloned into the pcDNA3.1 vector. The cloning was confirmed by PCR screening, restriction enzyme reactions, and DNA sequence analysis. Finally, three-dimensional structure and antigenic properties of the protein encoded by the LtTSA were determined.ResultsSix hundred base pair bands belonging to LtTSA were shown with electrophoresis. It was found that LtTSA and its encoded protein have high similarity with different Leishmania species. LtTSA protein consisting of 199 amino acids was found to have 7 different antigenic regions. ConclusionLtTSA and its encoded TSA protein were found to be highly immunogenic and similar to TSA proteins previously tested as a vaccine candidate.

Promastigote parasites cultured from the lesions of patients with mucosal leishmaniasis are more resistant to oxidative stress than promastigotes from a cutaneous lesion

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H2O2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment.

Overexpression of a peroxiredoxin gene from Tamarix hispida, ThPrx1, confers tolerance to oxidative stress in yeast and Arabidopsis

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (H2O2) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under H2O2 and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of H2O2, superoxide (O2•−) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress.

23 - Insights into the Mechanism of Peroxiredoxin 6 Sulfenic Acid reduction by Ascorbate

Human peroxiredoxin 6 (PRDX6) is the only mammalian 1-Cys based member of the Prx family.1 In the first step of its catalytic cycle a sulfenic acid (CP-SOH) is formed at the peroxidatic cysteine, C47, which is then reduced by a thiol-containing agent like glutathione or by ascorbate. The finding of ascorbate as a Prx reducing agent2 revolutionized the thiol-specific antioxidant paradigm, expanding the world of possible 1-Cys Prxs reductants. Rate constants for reduction of CP-SOH by ascorbate have been measured for different 1-Cys based Prxs at L. Netto’s Lab, obtaining values in the 500-2000 M-1s-1 range. Here we address the molecular mechanism of PRDX6 CP-SOH reduction by ascorbate from a theoretical perspective, by combining molecular dynamics and ligand-protein docking with QM/MM methods. Our results show the reaction follows a SN2-like substitution mechanism coupled at the transition state with a proton transfer (PT) from R155 into the leaving OH-. A free-energy activation barrier of 11.5 kcal-1mol-1 is obtained as our best theoretical estimation, which can be transformed into a rate constant of 2.2 x 104M-1 s-1 using the Eyring-Polanyi equation, in very good agreement with reported data3. This kind of mechanism represents a novelty for sulfenic acid reduction and will be discussed in detail, dissecting the role of PRDX6 residues critical in determining the reaction efficiency.

Nucleophosmin Regulates Intracellular Oxidative Stress Homeostasis via Antioxidant PRDX6.

Reactive oxygen species (ROS) play both deleterious and beneficial roles in cancer cells. Nucleophosmin (NPM) is heavily implicated in cancers of diverse origins, being its gene over-expression in solid tumors or frequent mutations in hematological malignancies. However, the role and regulatory mechanism of NPM in oxidative stress are unclear. Here, we found that NPM regulated the expression of peroxiredoxin 6 (PRDX6), a member of thiol-specific antioxidant protein family, consequently affected the level and distribution of ROS. Our data indicated that NPM knockdown caused the increase of ROS and its relocation from cytoplasm to nucleoplasm. In contrast, overexpression or cytoplasmic localization of NPM upregulated PRDX6, and decreased ROS. In addition, NPM knockdown decreased peroxiredoxin family proteins, including PRDX1, PRDX4, and PRDX6. Co-immunoprecipitation further confirmed the interaction between PRDX6 and NPM. Moreover, NSC348884, an inhibitor specifically targeting NPM oligomerization, decreased PRDX6 and significantly upregulated ROS. These observations demonstrated that the expression and localization of NPM affected the homeostatic balance of oxidative stress in tumor cells via PRDX6 protein. The regulation axis of NPM/PRDX/ROS may provide a novel therapeutic target for cancer treatment. J. Cell. Biochem. 118: 4697-4707, 2017. © 2017 Wiley Periodicals, Inc.

Immunological evidence for the presence of peroxiredoxin in pea leaf peroxisomes and response to oxidative stress conditions

Peroxiredoxins (Prxs) constitute a group of thiol-specific antioxidant enzymes which are present in bacteria, yeasts, and in plant and animal cells. Although Prxs are mainly localized in the cytosol, they are also present in mitochondria, chloroplasts, and nuclei, but there is no evidence of the existence of Prxs in plant peroxisomes. Using soluble fractions (matrices) of peroxisomes purified from leaves of pea (Pisum sativum L.) plants, the immunological analysis with affinity-purified IgG against yeast Prx1 revealed the presence of an immunoreactive band of about 50 kDa. The apparent molecular mass of the peroxisomal Prx was not sensitive to oxidizing and reducing conditions what could be a mechanism of protection against the oxidative environment existing in peroxisomes. Postembedment, EM immunocytochemical analysis with affinity-purified IgG against yeast Prx1 antibodies, confirmed that this protein was present in the peroxisomal matrix, mitochondria, and chloroplasts. In pea plants grown under oxidative stress conditions, the protein level of peroxisomal Prx was differentially modulated, being slightly induced by growth of plants with 50 µM CdCl2, but being significantly reduced by treatment with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The presence in the matrix of peroxisomes of a protein immunorelated to Prx of about 50 kDa, which is in the range of molecular mass of the dimeric form of other Prxs, opens new questions on the molecular properties of Prxs, but also on their function in the metabolism of reactive oxygen and nitrogen species (ROS/RNS) in these plant cell organelles, where they could be involved in the regulation of hydrogen peroxide and/or peroxynitrite.

Abstract 2807: Coordinated upregulation of peroxiredoxins in breast cancer

Abstract Peroxiredoxins (Prdxs) are a group of thiol-specific antioxidant proteins that are involved in many cellular processes, including growth, proliferation, differentiation, and the stress response. The family includes six proteins in humans, which show differences in substrate specificity, tissue expression, and cellar localization and distribution. Cancer cells commonly exhibit aberrantly regulated expression of antioxidants, which can support cancer cell survival in the face of elevated levels of reactive oxygen species. Breast cancers exhibit significantly elevated Prdx levels of Prdx. We previously reported overexpression of many Prdx proteins in the MCF-7 breast cancer line, and further demonstrated their importance in cell survival and doxorubicin-resistance in this line. To further investigate the misregulation of these proteins in human breast cancer, we compared Prdx levels between breast tumor and adjacent normal breast tissue from 20 breast cancer patients. We showed that most patients exhibited elevated levels of several Prdx proteins in the tumor tissue. We have gone on to analyze the promoters of these genes in an effort to identify potential mechanisms of coordinate regulation, as well as downstream targets that may mediate their role in cell survival. Together, we feel that this family of proteins may be useful biomarkers, and also targets, in breast cancer biology. Citation Format: Jillian Muhlbauer, Shelley A. Phelan. Coordinated upregulation of peroxiredoxins in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2807.

Prdx Overexpression in Tumor Tissue of Breast Cancer Patients

Peroxiredoxin (Prdx) proteins are evolutionarily conserved thiol-specific antioxidant enzymes that reduce various cellular peroxides, protecting cells from oxidative damage. Prdxs also have been demonstrated to play an important role in regulating redox-sensitive cell signaling in a number of cell processes.