[Comparative analysis of post-translational modifications in plasma proteome of patients with cerebral ischemia based on HPLC-MS/MS method]. / Sravnitel'nyi analiz modifikatsii proteoma plazmy krovi bol'nykh tserebral'noi ishemiei na osnove metoda VÉZhKh-MS/MS.

The relative differences between post-translational modifications (PTM) of proteins in blood plasma samples of patients with cerebral ischemia (CI) and healthy people were investigated using of the method of label-free comparative proteomic analysis based on the technology of tandem HPLC-MS/MS. For PTM detection we used multiple MS/MS search in the database Mascot for variable PTM and Progenesis LS-MS software. In the CI plasma samples, we observed an increase in the proportion of peptides with such PTM as phosphorylation of serine, threonine, and tyrosine, acetylation of lysine and protein N-term, ubiquitination of lysine and deamidation of glutamine related to clinically significant processes were revealed.

Analysis of Blood Plasma Protein Composition in Patients with Cerebral Ischemia.

Blood plasma proteome in patients with cerebral ischemia and healthy individuals was studied using comparative proteomic analysis based on tandem HPLC-MS/MS. Mass spectra were analysed in an automated mode using Progenesis LS-MS software and 256 proteins were identified. Significant quantitative differences were revealed for 20 proteins. It was found that changes in the blood plasma proteome in subjects with cerebral ischemia involved a wide range of proteins: molecular chaperones, fibrinolysis, angiogenesis, and immune system proteins, proteins involved in homeostasis maintenance, cell differentiation and proliferation, regulators of apoptosis, and cytoskeleton proteins.

[Comparative proteome analysis of blood plasma of patients with early-stage chronic cerebral ischemia]. / Sravnitel'nyi analiz proteoma plazmy krovi bol'nykh na rannei stadii khronicheskoi tserebral'noi ishemii.

In the present study, we explored the technology of liquid chromatography-mass spectrometry (HPLC-MS/MS) for the proteome analysis of blood plasma of patients with early chronic cerebral ischemia. Analysis of mass-spectrometer data carried out in automatic mode using the software Progenesis LS-MS. As a result of this study identified 43 proteins. The differences identified in the study group compared with the control in 7 proteins. It was found that in the early stages of chronic cerebral ischemia proteome changes in blood plasma affect proteins related to the immune system, the system for the maintenance of hemostasis and lipid metabolism.

[Analysis of proteomic profile changes of zebrafish embryos during exposure to doxorubicin, built-in the phospholipid transport nanosystem].

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.

Effects of fullerene C60 on proteomic profile of Danio rerio fish embryos.

The effects of phosphatidylcholine-based phospholipid nanoparticles containing fullerene C60 on Danio rerio fish embryos were studied. Exposure of the embryos with the nanoparticles for 48 h did not lead to appreciable changes in the number of protein bands in SDS-PAGE in comparison with the control (exposure in medium with phosphatidylcholine). Mass spectrometric identification of proteins showed differences in the proteomic profiles of the samples. The content of vitellogenins changed after exposure with phosphatidylcholine-based nanoparticles with C60 fullerenes. This could indicate low toxicity of the nanoparticles towards D. rerio embryos under experimental conditions.

[Study of proteomic profile of Danio rerio embryos using one-dimensional electrophoresis and mass spectrometry].

In the present study, a proteomic technology combining one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry (MALDI-TOF-PMF) has been successfully applied for revelation of changes in the protein profile of zebrafish (Danio rerio) 52 hpf embryos. Prior to 1DE separation of zebrafish embryonic proteins, the procedure for obtaining embryos homogenate was optimized by ultrasonic treatment. A total of 84 proteins, including 15 vitellogenins, were identified. It was shown that growing ofzebrafish embryos in the medium with doxorubicin (DOX) stimulated Caspase-3 induction and promoted the disappearance of cardiac troponins, both these findings being consistent with literature data on doxorubicin-induced cardiotoxicity. The 1DE-based proteomic mapping approach proposed herein enabled not only to identify proteins but also to register those changes in embryos' proteomic profile that were caused by doxorubicin.

Electrochemistry of cytochromes p450: analysis of current-voltage characteristics of electrodes with immobilized cytochromes p450 for the screening of substrates and inhibitors.

In the current study, an approach to elucidating the substrate specificity of cytochromes P450 based on the analysis of current-voltage characteristics of voltammograms and amperograms is proposed. Data on the electrochemical behavior of bioelectrodes with immobilized cytochromes P450 2B4, 1A2, 3A4, 11A1 (P450scc), and 51b1 (Mycobacterium tuberculosis sterol 14alpha-demethylase or CYP51 MT) in the presence of typical substrates and inhibitors for these hemoprotein forms are reported. Immobilization of the enzymes was accomplished by using graphite screen-printed electrodes modified with gold nanoparticles and with the synthetic membrane-like compound didodecyldimethylammonium bromide. The method of electro-analysis can be applied to the search of potential substrates and inhibitors of cytochromes P450 and to creation of multichannel electrochemical plates (chips, panels) with immobilized cytochromes P450.

Electrochemical reduction of sterol-14alpha-demethylase from Mycobacterium tuberculosis (CYP51b1).

The electrochemical reduction of the heme protein sterol-14alpha-demethylase from Mycobacterium tuberculosis (CYP51b1, or further CYP51) was investigated. Direct electron transfer was demonstrated between CYP51 and graphite screen-printed electrodes modified with gold nanoparticles and with the membrane-like synthetic surfactant didodecyl dimethylammonium bromide. The formal potential of the Fe3+/Fe2+ pair, E(1/2), is equal to -273 mV (vs. Ag/AgCl). The cathodic current corresponding to the reduction of oxygen by immobilized heme protein was registered in the presence of oxygen. Addition of lanosterol, one of the substrates of the CYP51 family, to the oxygenated solution caused a concentration-dependent increase in the reduction current in voltammetric and amperometric experiments. Ketoconazole, an inhibitor of CYP51, inhibited the catalytic cathodic current in the presence of lanosterol. Electrochemical reduction of CYP51 may serve as an adequate alternative to the reconstituted system, which requires additional redox partners for the exhibition of catalytic activity of heme proteins of the cytochrome P450 superfamily.

Electrochemical properties of cytochroms P450 using nanostructured electrodes: direct electron transfer and electro catalysis.

The present study demonstrates direct electron transfer between cytochromes P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51b1) on the one hand and screen-printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB) on the other. Electro detection of heme proteins was possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, and lanosterol) and with P450 inhibitor (ketoconazole) were analyzed using cyclic voltammetry (CV), square wave voltammetry (SWV) differential pulse voltammetry (DPV), and amperometry.

[Electrochemical reduction of cytochrome P450s based on electrodes with immobilized substrates].

A new approach for the electrochemical reduction of cytochromes P450 (P450s, CYPs) with electrodes chemically modified with appropriate substrates of P450s ("reverse" electrodes) has been proposed. The method is based on the analysis of cyclic voltammograms, square wave voltammograms, amperograms and determination of such electrochemical characteristics as catalytic current and redox potential. The sensitivity of the proposed method is 0.2-1 nmol P450/electrode. The differences of maximal current and potentials in square wave voltammograms and catalytic current in amperometric measurements are more sensitive and reliable. Planar regime of screen-printed electrodes permits to use 20-60 microl of electrolyte volume. We investigated P450 2B4--benzphetamine or P450scc--cholesterol enzyme - substrate pairs. Electrochemical parameters of electrodes with nonspecific P450 substrate were differed from electrodes with appropriate substrates.

[Study of interaction of cytochrome P450 2B4 with riboflavin by fluorescence spectrometry]. / Issledovanie vzaimodeistviia tsitokhroma P450 2B4 s riboflavinom metodami fluorestsentnoi spektroskopii.

Fluorescence quenching of riboflavin by cytochrome P450 2B4 was used to probe the ligand--enzyme binding interaction ((lambda ex = 385 nm, lambda em = 520 nm). Riboflavin is a component of a flavoprotein NADPH dependent cytochrome P450 reductase, an essential electron carrier during cytochrome P450 catalysis. Fluorescence titration measurements revealed that cytochrome P450 2B4 and riboflavin formed a complex with an apparent Kd = 8.8 +/- 1 microM. The fluorescence intensity of riboflavin decreased upon the addition of cytochrome P450 2B4, which may be caused by the resonance excitation energy transfer from the fluorescent donor riboflavin to the cytochrome P450 2B4 heme acceptor. These data suggest that there may exist specific sites of binding of riboflavin with the protein globule of cytochrome P450 2B4.

[Structural-functional motifs of sterol 14-alpha demethylases (CYP51)]. / Strukturno-funktsional'nye motivy sterolovykh 14-al'fa-demetilaz (CYP51).

CYP51 family of cytochromes P450 (sterol 14-alpha-demethylases) comprises the representatives from different kingdoms of living world, thus positioning itself as the most ancient member of the superfamily. In the course of the present research the collection of 36 full-length CYP51 amino acid sequences was submitted to cluster analysis. Each node of the clustering dendrogram corresponds to the groups of proteins, located on the branches descending from the node. By making the multiple alignment of each group of protein sequences we obtained the node-specific consensus sequences. The informational content of the consensus was defined as the presence of the compact conserved sites, the motifs. The assessment of informational content was computed using Sherman's non-parametric statistical criterion. The high informational content was observed for the 100% conserved consensus sequences of the following CYP51,s groups: fungi, animal+plant, plant+protista and bacteria. These selected consensus sequences were next aligned all together to get the final consensus for the whole family. To enrich the informational content of the CYP51 consensus the level of its conservation was dropped to 75%. Regions of statistically significant conservation were unraveled in the CYP51 consensus sequence. These regions (motifs) were then correlated with the information on secondary structure elements and substrate recognition sites reported for CYP51 from Mycobacterium tuberculosis. Seven motifs appeared to be obligatory for every CYP51 protein. The motifs thus obtained were searched for among all the known cytochrome P450 proteins. Some motifs were found to be absolutely specific for 14-alpha-demethylases, whereas others were common to different species of cytochromes P450.

Optical biosensor investigation of interactions of biomembrane and water-soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers.

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions.

Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system.

The mechanism of the cytochrome P450 2B4 modification by hydrogen peroxide (H2O2) formed as a result of partial coupling of NADPH-dependent monooxygenase reactions has been studied in the monooxygenase system reconstituted from the highly purified microsomal proteins: cytochrome P450 2B4 (P450) and NADPH-cytochrome P450 reductase in the presence of detergent Emulgen 913. It was found, that H2O2-mediated P450 self-inactivation during benzphetamine oxidation is accompanied by heme degradation and apoenzyme modification. The P450 heme modification involves the heme release from the enzyme under the action of H2O2 formed within P450s active center via the peroxycomplex decay. Additionally, the heme lost is destroyed by H2O2 localized outside of enzyme's active center. The modification of P450 apoenzyme includes protein aggregation that may be due to the change in the physico-chemical properties of the inactivated enzyme. The modified P450 changes the surface charge that is confirmed by the increasing retention time on the DEAE column. Oxidation of amino acid residues (at least cysteine) may lead to the alteration into the protein hydrophobicity. The appearance of the additional ionic and hydrophobic attractions may lead to the increase of the protein aggregation. Hydrogen peroxide can initiate formation of crosslinked P450 dimers, trimers, and even polymers, but the main role in this process plays nonspecific radical reactions. Evidence for the involvement of hydroxyl radical into the P450 crosslinking is carbonyl groups formation.

High-pressure-induced transitions in microsomal cytochrome P450 2B4 in solution: evidence for conformational inhomogeneity in the oligomers.

Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a function of benzphetamine concentration (0.05 divided by 2 mM) and state of aggregation of the hemoprotein in solution. Application of factor analysis to the spectral changes in the Soret region allowed us to resolve two particular pressure-induced processes in 2B4 oligomers. The first process was identified as the conversion of the low-spin P450 into the P420 state. At 25 degrees C it was followed by decay (bleaching) of about 50% of the newly formed P420. The second process was a pressure-induced high- to low-spin shift. Both transitions were reversible, except the hemoprotein bleaching. The amplitude of the P450-->P420 transition accounted for 67 +/- 5% of the total hemoprotein content. Furthermore, the fraction of the hemoprotein exposed to spin equilibrium was not affected by the P450-->P420 conversion and was estimated to be only about 31 +/- 5% of the total hemoprotein content. After the dissociation of the oligomers by 0.2% Triton N-101, the inhomogeneity vanished: 95% of the monomers were involved in the P450-->P420 transition (delta V degrees = -86 ml/mol) followed by intense bleaching of the hemoprotein. This agrees with our earlier observations on the reduced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers. The parameters of the P450-->P420 conversion (delta V degrees = -32 ml/mol, P1/2 = 1560 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus benzphetamine concentration shows this transition to be caused mainly by changes in the spin equilibrium of both substrate-bound (delta V degrees = -49 ml/mol) and substrate-free (delta V degrees = -21 ml/mol) hemoprotein, whereas the substrate binding step itself has a very weak pressure dependency (delta V degrees = -8 ml/mol).

Membrane topology of N-terminal residues of cytochromes P-450 2B4 and 1A2.

The paper describes the reactivity of fluorescein isothiocyanate towards the N-terminus of cytochromes P450 2B4 and 1A2 in solution, in the natural membrane of microsomes and in proteoliposomes (cholate and ultrasonic). The results obtained indicate, that the N-terminus of microsomal or proteoliposomal cytochromes P-450 2B4 and 1A2 spans the membrane only once and faces the vesicles interior. It was suggested that of major importance in orientation of N-terminal residues in the membrane is not the hydrophobic segment itself but rather the positively charged fragment, following it.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.

[The role of heme in formation of the native structure of cytochrome P-450 LM2]. / Rol' gema v formirovanii nativnoi struktury tsitokhroma P-450 LM2.

The role of heme in the formation of cytochrome P-450 native structure was investigated. It was shown that treatment of purified and membrane-bound hemoproteins with H2O2 results in the total destruction of heme. After incubation with hemine the apoprotein thus obtained forms a catalytically active cytochrome P-450. The efficiency of this process depends on the enzyme microenvironment. The membrane-bound apoprotein may be reconstituted by 70-80%, whereas the soluble one--by 50%. It is concluded that the observed differences may be accounted for by a greater stability of the membrane-bound protein structure.