HPLC with chiral stationary phase for separation and kinetics study of aspartic acid epimerization in Peroxiredoxin 2 active site peptide.

Amino acid epimerization, a process of converting L-amino acids to D-amino acids, will lead to modification in the protein structure and, subsequently, its biological function. This modification causes no change in protein m/z and may be overlooked during protein analysis. Aspartic Acid Epimerization (AAE) is faster than other amino acids and could be accelerated by free radicals and peroxides. In this work, a novel and site-specific HPLC method using a chiral stationary phase for determining the AAE in the active site model peptide (AP) of Peroxiredoxin 2 has been developed and validated. The developed method showed good linearity (1 - 200 µg/mL) and recoveries of the limit of quantification (LOQ), low, medium, and high concentrations were between 85% and 115%. The Kinetics of AAE in AP were studied using the developed method, and the results showed that when ascorbic acid and Cu2+ coexisted, the AP epimerized rapidly. The AAE extent increased with time and was positively correlated with hydrogen peroxide generation.

Systematic monitoring of 2-Cys peroxiredoxin-derived redox signals unveiled its role in attenuating carbon assimilation rate.

Transmission of reductive and oxidative cues from the photosynthetic electron transport chain to redox regulatory protein networks plays a crucial role in coordinating photosynthetic activities. The tight balance between these two signals dictates the cellular response to changing light conditions. While the role of reductive signals in activating chloroplast metabolism is well established, the role of their counterbalanced oxidative signals is still unclear, mainly due to monitoring difficulties. Here, we introduced chl-roGFP2-PrxΔCR, a 2-Cys peroxiredoxin-based biosensor, into Arabidopsis thaliana chloroplasts to monitor the dynamic changes in photosynthetically derived oxidative signaling. We showed that chl-roGFP2-PrxΔCR oxidation states reflected oxidation patterns similar to those of endogenous 2-Cys peroxiredoxin under varying light conditions. By employing a set of genetically encoded biosensors, we showed the induction of 2-Cys peroxiredoxin-dependent oxidative signals, throughout the day, under varying light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals. Furthermore, we demonstrated the induction of 2-Cys peroxiredoxin-derived oxidative signals during a dark­to­low-light transition and uncovered a faster increase in carbon assimilation rates during the photosynthesis induction phase in plants deficient in 2-Cys peroxiredoxins compared with wild type, suggesting the involvement of oxidative signals in attenuating photosynthesis. The presented data highlight the role of oxidative signals under nonstress conditions and suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system.

A Novel TLR4-Binding Domain of Peroxiredoxin From Entamoeba histolytica Triggers NLRP3 Inflammasome Activation in Macrophages.

Macrophages promote early host responses to infection by releasing pro-inflammatory cytokines, and they are crucial to combat amoebiasis, a disease affecting millions of people worldwide. Macrophages elicit pro-inflammatory responses following direct cell/cell interaction of Entamoeba histolytica, inducing NLRP3 inflammasome activation with high-output IL-1ß/IL-18 secretion. Here, we found that trophozoites could upregulate peroxiredoxins (Prx) expression and abundantly secrete Prxs when encountering host cells. The C-terminal of Prx was identified as the key functional domain in promoting NLRP3 inflammasome activation, and a recombinant C-terminal domain could act directly on macrophage. The Prxs derived from E. histolytica triggered toll-like receptor 4-dependent activation of NLRP3 inflammasome in a cell/cell contact-independent manner. Through genetic, immunoblotting or pharmacological inhibition methods, NLRP3 inflammasome activation was induced through caspase-1-dependent canonical pathway. Our data suggest that E. histolytica Prxs had stable and durable cell/cell contact-independent effects on macrophages following abundantly secretion during invasion, and the C-terminal of Prx was responsible for activating NLRP3 inflammasome in macrophages. This new alternative pathway may represent a potential novel therapeutic approach for amoebiasis, a global threat to millions.

PRDX1 gene-related epi-cblC disease is a common type of inborn error of cobalamin metabolism with mono- or bi-allelic MMACHC epimutations.

BACKGROUND: The role of epigenetics in inborn errors of metabolism (IEMs) is poorly investigated. Epigenetic changes can contribute to clinical heterogeneity of affected patients but could also be underestimated determining factors in the occurrence of IEMs. An epigenetic cause of IEMs has been recently described for the autosomal recessive methylmalonic aciduria and homocystinuria, cblC type (cblC disease), and it has been named epi-cblC. Epi-cblC has been reported in association with compound heterozygosity for a genetic variant and an epimutation at the MMACHC locus, which is secondary to a splicing variant (c.515-1G > T or c.515-2A > T) at the adjacent PRDX1 gene. Both these variants cause aberrant antisense transcription and cis-hypermethylation of the MMACHC gene promotor with subsequent silencing. Until now, only nine epi-cblC patients have been reported. METHODS: We report clinical/biochemical assessment, MMACHC/PRDX1 gene sequencing and genome-wide DNA methylation profiling in 11 cblC patients who had an inconclusive MMACHC gene testing. We also compare clinical phenotype of epi-cblC patients with that of canonical cblC patients. RESULTS: All patients turned out to have the epi-cblC disease. One patient had a bi-allelic MMACHC epimutation due to the homozygous PRDX1:c.515-1G > T variant transmitted by both parents. We found that the bi-allelic epimutation produces the complete silencing of MMACHC in the patient's fibroblasts. The remaining ten patients had a mono-allelic MMACHC epimutation, due to the heterozygous PRDX1:c.515-1G > T, in association with a mono-allelic MMACHC genetic variant. Epi-cblC disease has accounted for about 13% of cblC cases diagnosed by newborn screening in the Tuscany and Umbria regions since November 2001. Comparative analysis showed that clinical phenotype of epi-cblC patients is similar to that of canonical cblC patients. CONCLUSIONS: We provide evidence that epi-cblC is an underestimated cause of inborn errors of cobalamin metabolism and describe the first instance of epi-cblC due to a bi-allelic MMACHC epimutation. MMACHC epimutation/PRDX1 mutation analyses should be part of routine genetic testing for all patients presenting with a metabolic phenotype that combines methylmalonic aciduria and homocystinuria.

Inhibition of the antioxidant enzyme PRDX1 activity promotes MPP+-induced death in differentiated SH-SY5Y cells and may impair its colocalization with eEF1A2.

AIM: eEF1A2 is highly expressed in postmitotic cells and has been reported to interact with the antioxidant enzyme peroxiredoxin 1 (PRDX1). PRDX1 is involved in motor neuron differentiation. Here, we studied the relationship between eEF1A2 and PRDX1 during dopaminergic neuron differentiation, and examined their possible association in an oxidative stress model of Parkinson's disease (PD). MAIN METHODS: Expression of eEF1A2 and PRDX1 in SH-SY5Y cells at various durations of retinoic acid (RA) induction was detected using qRT-PCR, Western blotting and immunofluorescence. Neurons of 10-day differentiation were treated with the PRDX1 inhibitor H7, MPP+ and H7 plus MPP+. The cell viability, the amounts of apoptotic nuclei, DHE signals, and the expression of p53, p-Akt and p-mTOR were determined. The colocalization of eEF1A2 and PRDX1 was visualized using confocal microscopy. KEY FINDINGS: eEF1A2 gradually increased after RA-induced differentiation of SH-SY5Y cells, while PRDX1 protein gradually decreased. MPP+ treatment increased eEF1A2 in both undifferentiated and differentiated neurons; however, PRDX1 appeared to elevate only in mature neurons. The inhibition of the PRDX1 activity with H7 promoted MPP+-induced cell death, as evidenced by decreased cell viability, increased apoptotic nuclei, increased the DHE signal, and increased p53. However, H7 induced the activation of the prosurvival Akt and mTOR in MPP+-treated cells. Besides, a colocalization of eEF1A2 and PRDX1 was evidenced in MPP+-treated neurons. This colocalization was possibly prevented by inhibiting the PRDX1 activity, resulting in aggravated neuronal death. SIGNIFICANCE: Our results suggest that the possible association between eEF1A2 and PRDX1 may be a promising target for modifying neuronal death in PD.

PRDX2 plays an oncogenic role in esophageal squamous cell carcinoma via Wnt/ß-catenin and AKT pathways.

PURPOSE: To investigate the role of PRDX2 in esophageal carcinoma (ESCA). METHODS: The expression of PRDX2 was detected in ESCA tissues. And PRDX2 expression in two ESCA cell lines was knocked down. Cell proliferation, metastasis and invasion were detected in these cells. RESULTS: Here, we found that PRDX2 expression was significantly increased in ESCA tissues and was associated with a poor prognosis in ESCA patients. In addition, PRDX2 expression was significantly associated with pathological grading, infiltration degree and 5-year survival time in ESCA patients. Next, we knocked down PRDX2 expression by PRDX2-shRNA transfection in two ESCA cell lines, Eca-109 and TE-1. Proliferation analysis indicated that in vitro PRDX2 knockdown decreased growth and clone formation of ESCA cells. Scratch and transwell assays indicated that cell migration and invasion were significantly inhibited by PRDX2 knockdown. In addition, PRDX2 knockdown inhibited cell cycle of ESCA cells and down-regulated Cyclin D1-CDK4/6. Moreover, PRDX2 knockdown regulated proteins involved in mitochondrial-dependent apoptosis, including increased Bax and Caspase9/3 and decreased Bcl2. Mechanism investigation indicated that PRDX2 knockdown led to inactivation of Wnt/ß-catenin and AKT pathways. CONCLUSIONS: Our data suggest that PRDX2 may function as an oncogene in the development of ESCA via regulating Wnt/ß-catenin and AKT pathways. Our study fills a gap in the understanding of the role of PRDX2 in ESCA and provides a potential target for ESCA treatment.

Characterization of exosomes in peritoneal fluid of endometriosis patients.

OBJECTIVE: To demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo. DESIGN: Case-control experimental study. SETTING: Academic clinical center. PATIENT (S): Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total). INTERVENTION (S): None. MAIN OUTCOME MEASURE (S): Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes. RESULT (S): Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain. CONCLUSION (S): Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis.

Reactive oxygen species-regulating proteins peroxiredoxin 2 and thioredoxin, and glyceraldehyde-3-phosphate dehydrogenase are differentially abundant in induced sputum from smokers with lung cancer or asbestos exposure.

Lung cancer is a deadly disease, typically caused by known risk factors, such as tobacco smoke and asbestos exposure. By triggering cellular oxidative stress and altering the antioxidant pathways eliminating reactive oxygen species (ROS), tobacco smoke and asbestos predispose to cancer. Despite easily recognizable high-risk individuals, lung cancer screening and its early detection are hampered by poor diagnostic tools including the absence of proper biomarkers. This study aimed to recognize potential lung cancer biomarkers using induced sputum noninvasively collected from the lungs of individuals in risk of contracting lung cancer. Study groups composed of current and former smokers, who either were significantly asbestos exposed, had lung cancer, or were unexposed and asymptomatic. Screening of potential biomarkers was performed with 52, and five differentially abundant proteins, peroxiredoxin 2 (PRDX2), thioredoxin (TXN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), extracellular matrix protein 1 (ECM1), and protein S100 A8 (S100A8), were chosen to undergo validation, for their previously known connection with oxidative stress or cancer. Results from the validation in 123 sputa showed that PRDX2, TXN, and GAPDH were differentially abundant in sputa from individuals with lung cancer. TXN had a negative correlation with asbestos exposure, yet a positive correlation with smoking and lung cancer. Thus, tobacco smoking, asbestos exposure, and lung carcinogenesis may disturb the cellular redox state in different ways. A strong correlation was found among PRDX2, TXN, GAPDH, and S100A8, suggesting that these proteins may present a diagnostic biomarker panel to aid recognizing individuals at high risk of contracting lung cancer.

Linkage of typically cytosolic peroxidases to erythrocyte membrane - A possible mechanism of protection in Hereditary Spherocytosis.

Hereditary Spherocytosis (HS) is a non-immune hemolytic anemia associated to oxidative stress (OS), namely to the linkage of cytosolic antioxidant enzymes to the erythrocyte membrane. Our aims were to evaluate erythrocyte OS changes and the membrane linkage of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) in unsplenectomized (unspl) and splenectomized (spl) HS patients and to search for associations with clinical severity (in unspl HS patients). We studied 114 HS patients (74 unspl and 40 spl) and 30 healthy individuals and we evaluated membrane bound hemoglobin (MBH), membrane lipid-peroxidation (LPO), enzymatic activities of GPx and CAT and the amounts of membrane bound Prx2, GPx and CAT, as well as, clinical and analytical parameters for characterization of HS. We found that unspl HS patients showed clear signs of anemia and in spl HS, a correction to this anemia was observed; the latter patients presented higher levels of OS biomarkers, namely, MBH and LPO. CAT was detected in the membrane of all individuals (control and HS groups), while GPx and Prx2 were only present in HS patients; moreover, their linkage to the membrane (in HS) appears to be cumulative since membrane bound peroxidases amount was higher as the number of peroxidases detected increased. MBH increased with the number/amount of membrane bound peroxidases, however LPO levels remained similar. In conclusion, our data suggest that the binding of these typically cytosolic peroxidases to erythrocyte membrane may be part of a mechanism of membrane protection to maintain its integrity by possibly regulating LPO.

Peroxiredoxin-5 is a negative survival predictor in ovarian cancer.

OBJECTIVES: Peroxiredoxins (PRDXs) constitute a family of antioxidant enzymes which are also involved in the process of carcinogenesis. They are composed of six identified isoforms (PRDX-1-6) and are supposed to play different roles in tumor progression, depending on type of cancer and member of the PRDX family. The aim of the study was to assess the prog- nostic value of PRDXs in ovarian cancer. MATERIAL AND METHODS: a dataset of patients with ovarian cancer from The Cancer Genome Atlas was analyzed. Expression of PRDX-1 to 6 mRNA was evaluated in 260 samples. The prognostic value of PRDXs was assessed using the Cox regression model which included the following clinical and pathological data: age, clinical stage, tumor grade, and residual disease. RESULTS: Within the PRDXs family, only higher expression of PRDX-5 was associated with worse overall survival both, in unselected patients and > 50-year-olds. PRDX-5 expression and residual disease were independent negative prognostic factors of patient survival. CONCLUSIONS: PRDX-5 is a negative predictor of survival in ovarian cancer.

The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients.

PURPOSE: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. METHODS: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. RESULTS: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). CONCLUSION: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.

The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients

Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells.

Ca2+ entry through store-operated Ca2+ channels (SOCs) in the plasma membrane (PM) of hepatocytes plays a central role in the hormonal regulation of liver metabolism. SOCs are composed of Orai1, the channel pore protein, and STIM1, the activator protein, and are regulated by hormones and reactive oxygen species (ROS). In addition to Orai1, STIM1 also interacts with several other intracellular proteins. Most previous studies of the cellular functions of Orai1 and STIM1 have employed immortalised cells in culture expressing ectopic proteins tagged with a fluorescent polypeptide such as GFP. Little is known about the intracellular distributions of endogenous Orai1 and STIM1. The aims are to determine the intracellular distribution of endogenous Orai1 and STIM1 in hepatocytes and to identify novel STIM1 binding proteins. Subcellular fractions of rat liver were prepared by homogenisation and differential centrifugation. Orai1 and STIM1 were identified and quantified by western blot. Orai1 was found in the PM (0.03%), heavy (44%) and light (27%) microsomal fractions, and STIM1 in the PM (0.09%), and heavy (85%) and light (13%) microsomal fractions. Immunoprecipitation of STIM1 followed by LC/MS or western blot identified peroxiredoxin-4 (Prx-4) as a potential STIM1 binding protein. Prx-4 was found principally in the heavy microsomal fraction. Knockdown of Prx-4 using siRNA, or inhibition of Prx-4 using conoidin A, did not affect Ca2+ entry through SOCs but rendered SOCs susceptible to inhibition by H2O2. It is concluded that, in hepatocytes, a considerable proportion of endogenous Orai1 and STIM1 is located in the rough ER. In the rough ER, STIM1 interacts with Prx-4, and this interaction may contribute to the regulation by ROS of STIM1 and SOCs.

Peroxiredoxin-2 and zinc-alpha-2-glycoprotein as potentially combined novel salivary biomarkers for early detection of oral squamous cell carcinoma using proteomic approaches.

No effective screening method is available for oral squamous cell carcinoma (OSCC) that is recognized to influence by environmental factors as well as human papillomavirus (HPV) and Epstein-Barr virus (EBV). Therefore, we sought to identify salivary biomarkers for screening of OSCC with or without HPV and/or EBV infection. Saliva, lesion and oral exfoliated cells were collected from OSCC patients and cancer-free controls (CFCs) and grouped depending on their HPV- and EBV-infection status. Salivary protein was precipitated and subjected to 2-dimensional gel electrophoresis. Differential expression of proteins was identified by mass spectrometry and validated by Western blotting. Distinctive expression patterns of salivary proteins were detected in OSCC as compared with CFCs. Levels of peroxiredoxin-2 (PRDX-2) and zinc-alpha-2-glycoprotein (ZAG) were significantly up-regulated in OSCC cases (p<0.001) relative to CFCs. Similarly, these proteins were also up-regulated in lesion cells compared with oral exfoliated cells (p<0.001). However, the expression patterns of these proteins were not significantly influenced by patient histories (risk factors). In combination, these proteins yielded the highest discriminatory power (AUC=0.999), sensitivity (100%), and specificity (98.77%) in distinguishing the early stages of OSCC. The detection of PRDX-2 combining with ZAG protein could potentially be used as salivary biomarkers for early screening of OSCC. SIGNIFICANCE: Our findings demonstrate a useful of combined detection of PRDX-2 and ZAG as a salivary biomarker for the early detection of OSCC.

OsPRX2 contributes to stomatal closure and improves potassium deficiency tolerance in rice.

Peroxiredoxins (Prxs) which are thiol-based peroxidases have been implicated in the toxic reduction and intracellular concentration regulation of hydrogen peroxide. In Arabidopsis thaliana At2-CysPrxB (At5g06290) has been demonstrated to be essential in maintaining the water-water cycle for proper H2O2 scavenging. Although the mechanisms of 2-Cys Prxs have been extensively studied in Arabidopsis thaliana, the function of 2-Cys Prxs in rice is unclear. In this study, a rice homologue gene of At2-CysPrxB, OsPRX2 was investigated aiming to characterize the effect of 2-Cys Prxs on the K+-deficiency tolerance in rice. We found that OsPRX2 was localized in the chloroplast. Overexpressed OsPRX2 causes the stomatal closing and K+-deficiency tolerance increasing, while knockout of OsPRX2 lead to serious defects in leaves phenotype and the stomatal opening under the K+-deficiency tolerance. Detection of K+ accumulation, antioxidant activity of transgenic plants under the starvation of potassium, further confirmed that OsPRX2 is a potential target for engineering plants with improved potassium deficiency tolerance.

Spectrophotometric assays for measuring redox biomarkers in blood and tissues: the NADPH network.

Nicotinamide adenine dinucleotide (NAD+/NADH) along with its phosphorylated form (NADP+/NADPH) are two molecules ubiquitously present in all organisms, and they play key roles as cofactors in fundamental catabolic and anabolic processes, respectively. The oxidation of NADPH to NADP+ initiates a cascade of reactions, where a network of molecules is implicated. The molecules of this cascade form a network with eminent translational potential in redox metabolism. A special point of interest is that spectrophotometric assays have been developed both for NADH/NADPH and the molecules directly regulated by them. Therefore, crucial molecules of the NADPH-dependent redox network can be measured, and the results can be used to assess the bioenergetic and/or oxidative stress status. The main aim of this review is to collectively present the NADPH-related molecules, namely NADPH, NADH, NAD+ kinase, NADPH oxidase, peroxiredoxin, thioredoxin, thioredoxin reductase, and nitric oxide synthase, that can be measured in blood and tissues with the use of a spectrophotometer, which is probably the most simple, inexpensive and widely used tool in biochemistry. We are providing the researchers with reliable and valid spectrophotometric assays for the measurement of the most important biomarkers of the NADPH network in blood and other tissues, thus allowing the opportunity to follow the redox changes in response to a stimulus.

Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells.

Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis.

Alterations in NO/ROS ratio and expression of Trx1 and Prdx2 in isoproterenol-induced cardiac hypertrophy.

The development of cardiac hypertrophy is a complicated process, which undergoes a transition from compensatory hypertrophy to heart failure, and the identification of new biomarkers and targets for this disease is greatly needed. Here we investigated the development of isoproterenol (ISO)-induced cardiac hypertrophy in an in vitro experimental model. After the induction of hypertrophy with ISO treatment in H9c2 cells, cell surface area, cell viability, cellular reactive oxygen species (ROS), and nitric oxide (NO) levels were tested. Our data showed that the cell viability, mitochondrial membrane potential, and NO/ROS balance varied during the development of cardiac hypertrophy in H9c2 cells. It was also found that the expression of thioredoxin1 (Trx1) and peroxiredoxin2 (Prdx2) was decreased during the cardiac hypertrophy of H9c2 cells. These results suggest a critical role for Trx1 and Prdx2 in the cardiac hypertrophy of H9c2 cells and in the transition from compensated hypertrophy to de-compensated hypertrophy in H9c2 cells, and our findings may have important implications for the management of this disease.

Nrf2-peroxiredoxin I axis in polymorphous adenocarcinoma is associated with low matrix metalloproteinase 2 level.

Polymorphous adenocarcinoma (PAC) is a malignant epithelial neoplasm that affects almost exclusively the minor salivary glands, generally described as having a relatively good prognosis. Aberrant nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) activation in tumor cells has been associated with induction of antioxidant enzymes, such as peroxiredoxin I (Prx I) and increased matrix metalloproteinase (MMP) expression. In this context, the aim of the present study was to evaluate the expression of Nrf2 and correlate it with Prx I and MMP-2 secretion in PAC. Thirty-one cases of PAC from oral biopsies were selected and immunohistochemically analyzed for Nrf2 and Prx I. MMP-2 quantification was performed on primary cell cultures derived from PAC. Oral squamous cell carcinoma (OSCC) cell cultures were used as control. A high immunoexpression of Nrf2 was observed in both the cytoplasm and the nucleus of neoplastic cells from PAC. Nuclear staining for Nrf2 suggested its activation in the majority of the PAC cells, which was confirmed by the high expression of its target gene, Prx I. Quantification of MMP-2 secretion showed lower levels in PAC cell cultures when compared to OSCC cell cultures (p < 0.05). In conclusion, although Nrf2 overexpression has been frequently associated with high-grade malignancies, such relationship is not infallible and, in fact, the opposite may occur in low-grade tumors, such as PAC of minor salivary glands.

Analysis of Pelagia noctiluca proteome Reveals a Red Fluorescent Protein, a Zinc Metalloproteinase and a Peroxiredoxin.

Pelagia noctiluca is the most venomous jellyfish in the Mediterranean Sea where it forms dense blooms. Although there is several published research on this species, until now none of the works has been focused on a complete protein profile of the all body constituents of this organism. Here, we have performed a detailed proteomics characterization of the major protein components expressed by P. noctiluca. With that aim, we have considered the study of jellyfish proteins involved in defense, body constituents and metabolism, and furthered explore the significance and potential application of such bioactive molecules. P. noctiluca body proteins were separated by1D SDS-PAGE and 2DE followed by characterization by nanoLC-MS/MS and MALDI-TOF/TOF techniques. Altogether, both methods revealed 68 different proteins, including a Zinc Metalloproteinase, a Red Fluorescent Protein (RFP) and a Peroxiredoxin. These three proteins were identified for the first time in P. noctiluca. Zinc Metalloproteinase was previously reported in the venom of other jellyfish species. Besides the proteins described above, the other 65 proteins found in P. noctiluca body content were identified and associated with its clinical significance. Among all the proteins identified in this work we highlight: Zinc metalloproteinase, which has a ShK toxin domain and therefore should be implicated in the sting toxicity of P. noctiluca.; the RFP which are a very important family of proteins due to its possible application as molecular markers; and last but not least the discovery of a Peroxiredoxin in this organism makes it a new natural resource of antioxidant and anti-UV radiation agents.