Aleksandra Rodacka

Antioxidant Properties of Resveratrol and its Protective Effects in Neurodegenerative Diseases

Summary Resveratrol is a natural organic compound, polyphenol, produced naturally by some plants in response to several harmful factors such as attack by pathogens, UV radiation, or increased oxidative stress. Many experiments suggest that it triggers mechanisms that counteract aging-related effects and plays a role in insulin resistance as well. It also possesses beneficial properties such as anti-cancer, anti-inflammatory, blood-sugar-lowering and cardiovascular effects. It is supposed to exhibit an interesting activity in neuroprotection - mainly through activation of sirtuins and counteraction in forming peptide aggregates. Still research is needed to evaluate exactly how resveratrol protects neurons, and to develop new, potential, therapeutic drugs

Can melatonin delay oxidative damage of human erythrocytes during prolonged incubation

PURPOSE Melatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation. MATERIAL/METHODS Human erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37ºC either in absence or presence of melatonin at concentration range 0.02 mM-3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO₂, GSH, fluorescent label DCFH₂) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis. RESULTS The prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO₂ and DCFH₂ was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%•h⁻¹) was 50% lower than that of the control while the HbO₂ oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage. CONCLUSIONS Probably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO₂ and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.

[The biological significance of oxidative modifications of cysteine residues in proteins illustrated with the example of glyceraldehyde-3-phosphate dehydrogenase].

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key redox-sensitive protein, the activity of which is largely affected by oxidative modifications at its highly reactive cysteine residue in the active site of the enzyme (Cys-152). These modifications occur as a result of S-thiolation, S-nitrosylation or disulfide bonds that lead to aggregate formation. The oxidative changes not only affect the glycolytic function but also stimulate the participation of GAPDH in numerous cellular processes. In this review we describe how thiol modification of Cys-152 in GAPDH re-routes metabolic pathways in the cell and converts a metabolic enzyme into a pro-apoptotic factor. Especially interesting issue is the participation of GAPDH in the regulation of expression of endothelin 1 and nitrosylation of nuclear proteins. In the last section we describe involvement of GAPDH in the processes associated with neurodegenerative diseases.

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme’s active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer’s disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

The effect of radiation-induced reactive oxygen species (ROS) on the structural and functional properties of yeast alcohol dehydrogenase (YADH).

Abstract Purpose: To determine the mechanism underlying oxidative modifications caused by radiation-induced reactive oxygen species (ROS) and elucidate their effect on the structure and function of yeast alcohol dehydrogenase (YADH), a zinc-containing protein. Materials and methods: YADH was exposed to water radiolysis products in an air atmosphere. YADH oxidation products were determined by spectrophotometric and spectrofluorimetric methods. The extent to which oxidative modifications affected enzyme activity was also studied. Results: Water radiolysis products oxidized thiol groups leading to the release of zinc ions and the destruction of tryptophan and tyrosine residues. Those processes were accompanied by alterations in protein structure such as increased surface hydrophobicity, greater tryptophan accessibility to acrylamide, and changes in the secondary structure. Structural modifications were correlated with lower enzyme activity. Conclusion: During the process of functional and structural changes in YADH exposed to reactive oxygen species, a key part is the oxidation of cysteine residues attached to zinc and the release of zinc ions from the molecule. It may be assumed that ROS induce similar changes in many other zinc-containing proteins.

The role of resveratrol and melatonin in the nitric oxide and its oxidation products mediated functional and structural modifications of two glycolytic enzymes: GAPDH and LDH

Abstract Background Nitric oxide is a well-known gaseous signaling molecule and protein modifying agent. However, at higher concentrations or during oxidative stress nitric oxide may exert some deleterious effects on protein structure and function. Here we investigated the influence of nitric oxide and products of its oxidation on two glycolytic enzymes: GAPDH and LDH under in vitro nitrosative stress conditions. Secondly, we applied natural antioxidants: melatonin and resveratrol to examine their effects on the enzymes under studied conditions. Methods By means of UV–VIS and fluorescence spectroscopy methods we compared nitric oxide mediated changes of enzyme activities, amount of free sulfhydryl groups (-SH) and bis-ANS probe binding. Finally, we predicted potential cysteine residues modified by nitric oxide in studied proteins using GPS-SNO software. Results Our results indicated that nitric oxide reversibly inactivates GAPDH but does not affect the activity of LDH. Nitric oxide dependent GAPDH activity decline was accompanied by the reduction of the amount of free –SH groups and GAPDH-bound bis-ANS fluorescence. Reduction of the number of free –SH groups and protein-bound bis-ANS fluorescence was also observed in LDH treated with NO. Applied antioxidants increased inactivation of GAPDH and structural changes of GAPDH and LDH. Conclusions Nitric oxide modifies function and structure of thiol-dependent enzyme such as GAPDH and structure of LDH which function do not rely on cysteine thiols. Both resveratrol and melatonin exerted prooxidative properties in studied conditions. General significance Extensively studied antioxidants: resveratrol and melatonin may function as a prooxidative species under in vitro nitrosative stress conditions.