M. G. Sharapov

The Cloning, Expression, and Comparative Analysis of Peroxiredoxin 6 from Various Sources

Human, rat, Xenopus, and Drosophila (DPx2540 and DPx6005) peroxiredoxin cDNAs were cloned and expressed in Escherichia coli. The recombinant enzymes were compared with respect to enzymatic activity toward various substrates and protection of plasmid DNA from the Fenton reaction products. The activity toward H2O2 decreased in the following order: DPx2540 > human Prx6 > Xenopus Prx6 > rat Prx6 > DPx6005. The activity toward tret-butyl hydroperoxide decreased in the following order: DPx2540 = DPx6005 > rat Prx6 > Xenopus Prx6 > human Prx6. The efficiency of plasmid DNA protection from oxidative damage mediated by the Fenton reaction decreased in the order of DPx2540 > DPx6005 = rat Prx6 = human Prx6 > Xenopus Prx6. The optimal temperature for activity of all enzymes was 37°C. Peroxiredoxins from rat, Xenopus, and Drosophila (DPx6005) retained no less than 50% of their activity in a wider temperature range (10–50°C) as compared with the human and Drosophila (DPx2540) enzymes (25–45°C). The thermostability of the enzymes decreased in the following order: DPx6005 = rat > human > Xenopus > DPx2540. The results confirmed a negative correlation between the activity and stability of peroxiredoxin 6, especially in the case of the Xenopus and Drosophila enzymes.

Apomyoglobin Stability As Dependent on Urea Concentration and Temperature at Two pH Values

Equilibrium unfolding of apomyoglobin (ApoMb) in the presence of urea was studied as dependent on the temperature (5–2°C) at two pH values (5.7 and 6.2). Thermodynamic parameters of ApoMb transition from the native to the unfolded state were estimated under various conditions. Conformational changes in ApoMb were detected by tryptophan fluorescence and far-UV circular dichroism. The ApoMb stability and the cooperativity of its unfolding at 5°C were considerably lower than at other temperatures at both pH values, where ApoMb is in the native conformation.

Peroxiredoxins as multifunctional enzymes

Peroxiredoxins are an evolutionary ancient widespread group of selenium-independent peroxidases. Peroxiredoxins protect cells from various peroxides, play an important role in maintaining redox homeostasis, and are additionally involved in transmitting extracellular and intracellular signals. The review considers peroxiredoxins from different kingdoms of living organisms and discusses the recent data on their structure, function, and the expression regulation of their genes.

Modified peroxiredoxins as prototypes of drugs with powerful antioxidant action

The possibility of using modified peroxiredoxins as powerful antioxidant agents has been considered. Peroxiredoxins immobilized on perfluorocarbon emulsions and PTD-modified peroxiredoxins have been studied. It has been shown that peroxiredoxins efficiently bind to particles of perfluorocarbon emulsions, while maintaining their antioxidant properties. A panel of PTD-modified peroxiredoxins has been created and peroxiredoxins most effective both in antioxidant properties and the ability to penetrate cells have been selected. The modified peroxiredoxins obtained may serve as the basis for the design of drug with powerful antioxidant action.

The role of peroxiredoxin 6 in neutralization of X-ray mediated oxidative stress: effects on gene expression, preservation of radiosensitive tissues and postradiation survival of animals

Abstract Peroxiredoxins are redox-sensing multifunctional enzymes, among them peroxiredoxin 6 (Prx6) can neutralize the most broadest range of hydroperoxides and play an important role in maintaining the redox homeostasis of the cell. In the present study, radioprotective and signaling regulatory effects of Prx6 were demonstrated and characterized. Intravenously administered exogenous Prx6 protects the organism of mice from the destructive action of ionizing radiation in the lethal dose range of 5–10 Gy. Dose reduction factor of 1.4 Prx6 injection reduces the severity of radiation-induced leuko- and thrombopenia in irradiated animals, also preventing the destruction of epithelial cells in the small intestine. Injecting exogenous Prx6 also as its mutated form of Prx6–C47S lacking peroxidase activity affects the expression of genes involved in antioxidant response, DNA reparation, apoptosis and inflammatory processes, in bone marrow cells both in intact animals and in those subjected to ionizing radiation. The radioprotective properties of Prx6 are based, on the one hand, on the capability for ROS neutralization, and on the other hand – on the potentiality for activation of reparation processes of the cell under oxidative stress conditions. Prx6 can be considered as a potentially perspective radioprotective agent for the reduction of risks from the damaging action of ionizing radiation on the mammalian organism.

Changes in gene expression and titin (connectin) content in striated muscles of chronically alcoholized rats

Using the real-time reverse-transcription polymerase chain reaction (RT-PCR) and low percentage SDS-gel electrophoresis, changes in the gene expression and isoform composition of a giant sarcomeric protein titin (connectin) in cardiac muscle and changes in its isoform composition in skeletal muscle m. soleus of chronically alcoholized rats were studied. The decreased content of titin protein in examined muscles and decreases in titin gene expression in the myocardium of chronically alcoholized rats were found, which indicates the development of the pathological process.

Two isoforms of peroxiredoxin 6 of Xenopus laevis

The genome of Xenopus laevis codes for two genes of peroxiredoxin 6, i.e., xen1 (Acc. no. EMBL Data Bank-BCO54278) and xen2 (Acc. no. EMBL Data Bank-BCO54309). Both the genes were cloned and expressed in Escherichia coli. The amino acid sequences of Xen1 and Xen2 enzymes are identical by 95%, and they possess the same peroxidase activity as well as similar optimums of temperature, pH, and thermostability. The genes of peroxiredoxin 6 of Xenopus laevis considerably differ in the period of expression during ontogenesis; i.e., xen2 is expressed during every stage of development, somewhat more intensively after stages 0–5; the expression of xen1 is initiated later, i.e., during the developmental stages of 47–48 h. Expression of xen2 increases after the incubation of embryos in a medium with hydrogen peroxide. Comparison of the amino acid sequences of Xen1 and Xen2 proteins shows that only Xen2 can possess phospholipase activity because its amino acid sequence contain residues of the phospholipase A2 active center: Ser31, His25, and Asp139.

The Role of Intermolecular Disulfide Bonds in Stabilizing the Structure of Peroxiredoxins

The comparative characterization of thermal stability of human peroxiredoxins 1–6 (Prx1–Prx6) has been performed by physicochemical and biochemical methods and the role of disulfide bonds in stabilizing their structure has been shown. Prx1 and Prx2 among the tested peroxiredoxins exhibit the highest peroxidase activity and thermal stability. Prx1 and Prx2 are more than 2 times more active on average with H2O2 and tert-butyl hydroperoxide as substrates compared to other peroxiredoxins and retain at least 50% activity after 30 min heating at a temperature of 64°C, which is more than 10°C higher compared to Prx3–Prx6. The reduction of the disulfide bonds between Prx1 and Prx2 leads to a decrease of their thermal stability, comparable to the thermal stability of Prx3–Prx6, which confirms the important role of the intermolecular S–S bonds in stabilizing the structure of these proteins.

The Effect of Exogenous Peroxiredoxin 6 on the State of Mesenteric Vessels and the Small Intestine in Ischemia–Reperfusion Injury

Oxidative stress is the main component of pathogenesis in ischemia–reperfusion injury. The administration of exogenous antioxidants suppresses oxidative stress and may decrease the severity of ischemia–reperfusion injury. The intestine is one of the most sensitive organs to the effect of ischemia–reperfusion. A rat model of a small intestine ischemia–reperfusion injury, based on occlusion of the superior mesenteric artery, was used in this work. Recombinant peroxiredoxin 6, a representative of an ancient family of peroxidases that are able to neutralize a broad range of both organic and inorganic peroxides, was used as an exogenous antioxidant. The intravenous administration of the exogenous peroxiredoxin 6 prior to ischemia–reperfusion minimizes tissue injury and reduces apoptotic cell death in the intestine and the mesenteric vessels. The impact of the exogenous peroxiredoxin 6 upon the NO level elevation in animal blood has been shown to be correlated with the enhanced inducible NO synthase expression. Thus, the use of exogenous peroxiredoxin 6 in ischemia–reperfusion injury of the intestine and the mesenteric vessels promotes normalization of the tissue redox homeostasis, structure protection, and restoration of the microvasculature.

Construction of a fusion enzyme exhibiting superoxide dismutase and peroxidase activity

A chimeric gene construct encoding human peroxiredoxin 6 and Mn-superoxide dismutase from Escherichia coli was developed. Conditions for expression of the fusion protein in E. coli cell were optimized. Fusing of the enzymes into a single polypeptide chain with peroxiredoxin 6 at the N-terminus (PSH) did not affect their activities. On the contrary, the chimeric protein with reverse order of enzymes (SPH) was not obtained in a water-soluble active form. The active chimeric protein (PSH) exhibiting both peroxidase and superoxide dismutase activities was prepared and its physicochemical properties were characterized.