[Influence of N-alkynylaminosteroids on mitochondria function and autophagy in glioma cells]. / Vliianie N-alkinilaminosteroidov na funktsionirovanie mitokhondrii i protsessy autofagii v kletkakh gliomy.

In this work we examined the synthesized N-alkynyl-17-aminosteroids and N-alkynyl-20-aminosteroids (based on dehydroepiandrosterone and pregnenolone, respectively) for their effect on C6 rat glioma cell functions. At 10 µM, the compounds had an insignificant effect on C6 glioma mitochondrial membrane potential, but increased cell autophagy by 70-90%, comparable to the known autophagy inducer dexamethasone. Docking simulations predict a potential high-affinity interaction between N-alkynylaminosteroids and Keap1 and the Hedgehog pathway protein, Smoothened, which are involved in autophagy regulation. The possible mechanisms of observed processes are discussed.

[In silico modeling of izoniazid-steroid conjugates interactions with cytochromes P450 of mycobacteria and their bioconversion in vitro by the cells]. / In silico modelirovanie vzaimodeistviia kon"iugatov izoniazid-steroid s tsitokhromami P450 mikobakterii i ikh prevrashchenie in vitro v kletkakh dannykh mikroorganizmov.

Molecular docking of four hydrazones of isoniazid with steroids (dehydroepiandrosterone, pregnenolone, 16α,17α-epoxypregnenolone, cholestenone) - IDHEA, IPRE, IEP5, ICHN, to mycobacterial cytochromes P450 was performed. The in silico study has shown than these hydrazones can be effectively bound to CYP121, CYP124, CYP125, CYP126A1, CYP130, and CYP51 with binding energy ranged from -9 kcal/mol to -12 kcal/mol. Calculations also demonstrated enhancement of passive lipid bilayer permeability with respect to isoniazid. In vitro IDHEA, IPRE, IEPR were found to undergo bioconversion into their 3-keto-4-en derivatives. This suggests their ability to penetrate into M. tuberculosis H37Rv cells. The results of this study are important in the context of understanding of specificity of binding of synthetic steroid derivatives to mycobacterial CYPs and indicate the possibility of using the steroid compounds studied by us as new ligands for these enzymes.

[Synthesis and evaluation of N-alkynylaminosteroids as potential CYP450 17A1 inhibitors]. / N-alkinilaminosteroidy v kachestve potentsial'nykh ingibitorov tsitokhroma P450 17A1.

Four isomeric dehydroepiandrosterone- and pregnenolone-based N-alkynylaminosteroids were synthesized and tested in vitro for inhibition of heterologously expressed CYP17A1. The highest inhibitory activity was observed when the optimal number of side chain atoms was met. The conjugate based on pregnenolone containing an N-propynyl moiety was found to interefere with enzymatic activity most effectively and consistently in the micromolar range.

[Extraction effect of ultrasound during plasma clot disruption].

The products of plasma clot destruction by the low-frequency ultrasound (US) were analyzed by the combination of SDS gel-electrophoresis, gel filtration chromatography and scanning electron microscopy. It was found that US (27 kHz) did not cause activation of the plasmin system or covalent bonds cleavage in the fibrin molecules. With the US intensities less than 21.6 W/cm2 the extraction of blood serum proteins, which are located in the pores of the fibrin network was occurred. An increase of the intensity of ultrasonic action led to protofibril dissociation, which was accompanied by further release into the solution of the blood serum proteins located inside the fibrin fibers. Being extracted from aggregated plasma clot proteins free protofibrils formed insoluble fibrin particles.

From structure and functions of steroidogenic enzymes to new technologies of gene engineering.

Abstract (3/4) This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.

[Range of substrates and steroid bioconversion reactions performed by recombinant microorganisms Saccharomyces cerevisiae and Yarrowia lipolytica expressing cytochrome P450c17].

The relationship between 17alpha-hydroxylation and 20-oxidation-reduction of progesterone and some of its derivatives was studied in yeast strains Saccharomyces cerevisiae YEp51alpha, Yarrowia lipolytica E129A15, and expressing cytochrome P450c17. The key metabolites were found to be 17alpha-hydroxyprogesterone and 17alpha,20(alpha,beta)-dihydroxypregn-4-ene-3-ones. The bioconversion pathways of pregn-4-ene-20(alpha,beta)-ol-3-ones were determined. They included cycles of 20-oxidation, 17alpha-hydroxylation, and stereospecific 20-reduction. The efficiency and kinetic parameters of steroid bioconversion by the recombinant strains were determined. The role of yeast analogs of mammalian steroid dehydrogenases is discussed. It was found that any of the desired derivatives, 17alpha-hydroxyprogesterone or progesterone 17alpha,20(alpha,beta)-diols, could be obtained from progesterone.

Expression of cytochrome P450scc in Escherichia coli cells: intracellular location and interaction with bacterial redox proteins.

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.

[Effect of steroid biosynthesis modificators on the progesterone biotransformation by a recombinant yeasts expressing cytochrome P450c17].

Using the recombinant microorganisms S. cerevisiae GRF18 YEp5117alpha, expressing cytochrome P450c17 from bovine adrenal cortex, we investigated the influence of the various modificators of steroids biosynthesis on the relationship between the 17alpha-hydroxylation of progesterone and 20alpha-reduction. Dexamethasone and metirapon had no effect on the reaction of progesterone 17alpha-hydroxylation and on the reaction of 17alpha-hydroxyprogesterone 20alpha-reduction. Mifepriston and danazol did not covalently modify amino acid residues of the cytochrome P450c17 or its heme group under the conditions of the biotransformation of progesterone by recombinant yeasts. Ketokonazol, mifepriston and danazol acted as low-affinity competitive inhibitors, but the 20-dihydro derivatives of progesterone were mixed type inhibitors for the cytochrome P450c17. All modifiers that we used did not influence the functional properties of the yeast analog of 20alpha-hydroxysteroid dehydrogenase. According to the influence on the catalytic parameters of the cytochrome P450c17, the modifiers used can be arranged in the following order: 20beta-dihydroprogesterone (maximum effect) > mifepriston = ketokonazol > 20alpha-dihydroprogesteron > danazol > dexamethasone, metirapon (without effect).

Effect of ultrasound on activation of serine proteases precursors.

The effect of ultrasound (US) (26.4 kHz, 26 W/cm2) on the activation process of a mixture of chymotrypsinogen and trypsinogen was studied. US led to a significant decrease in proteolytic activity, as well as inhibition of the activation process in general. It was shown that inhibition of proteinase activity under US influence was a consequence of inhibition of chymotrypsinogen-chymotrypsin transformation and the complete proteolytic trypsin degradation in the proenzymes mixture.

Effect of ultrasound on structure and functional properties of antithrombin III and proteins of PPSB complex.

A combination of gel-permeation HPLC, affinity chromatography on heparin-Sepharose, gel electrophoresis, and estimation of inhibitory activity showed that effect of low-frequency ultrasound (26 W/cm(2), 37 degrees C, pH 7.4) on homogeneous antithrombin III was accompanied by formation of aggregates and a latent form of serpin. Heparin and pentosan polysulfate stabilized antithrombin III; this resulted in decrease in ultrasonic-induced formation of the aggregate and latent forms. The influence of ultrasound was not accompanied by significant changes in the contents of non-activated blood coagulation factors in the PPSB complex.

[Structure-functional change in streptokinase exposed to ultrasound]. / Strukturno-funktsional'nye izmeneniia streptokinazy pod deistviem ul'trazvuka.

To optimize conditions of acousto-enzymatic thrombolysis the influence of low-frequency ultrasound on the isolated preparation of streptokinase was investigated. The ultrasound treatment with intensity 26 W/cm2 at 37 degrees C within 5-10 minutes was not accompanied by changes of structure-functional properties of the streptokinase molecule. Increase of ultrasound-processing time (10-60 minutes) resulted in non-covalent hydrophobic aggregation of some part of the protein. In contrast to native protein ultrasound modified streptokinase is readily degraded by plasmin with formation of polypeptide fragments with molecular weights ranged from 43 up to 14 kD. The processes of aggregation and increased proteolytic degradation resulted in lower efficiency of plasmin autoactivation under the action of sounded streptokinase.

[Oxidation of 17alpha,20beta- and 17alpha,20alpha-dihydroxysipregn-4-en-ones--side products of biotransformation of progesterone by recombinant microorganisms expressing cytochrome P45017alpha]. / Okislenie 17alpha,20beta- i 17alpha,20alpha-digidroksipregn-4-en-3-onov--pobochnykh produktov biotransformatsii progesterona rekombinantnymi mikroorganizmami, ékspressiruiushchimi tsitokhorm P45017alpha.

Progesterone biotransformation with recombinant yeast Yarrowia lipolytica E129A15 and Saccharomyces cerevisiae GRF18/YEp5117 alpha expressing bovine adrenocortical cytochrome P45017 alpha yielded 17 alpha-hydroxyprogesterone and two diols, 17 alpha, 20 beta- and 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one. The oxidation of mixtures of the three steroids with chromic acid resulted in the cleavage of 17-20 bonds in the diols with the formation of androst-4-ene-3,17-dione. The biotransformation of pregn-4-ene-20 beta-ol-3-one by means of Y. lipolytica E129A15 was accompanied by the following reactions: the primary oxidation of these compounds to progesterone and the subsequent successive reactions of 17 alpha-hydroxylation and 20 alpha- and 20 beta-reduction. The results widen the possibilities for enzymatic and chemical modifications of steroids. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

Biotransformation of steroids by a recombinant yeast strain expressing bovine cytochrome P-45017alpha.

The cDNA encoding cytochrome P-45017alpha from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactose-induced yeast cells showed expression of about 240 nmol of P-45017alpha per liter of the culture. Binding of progesterone to the cytochrome P-45017alpha was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17alpha-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017alpha and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17alpha-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017alpha-dependent hydroxylation reaction. The reduction of the 20-keto group of 17alpha-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017alpha expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11beta- > 11alpha- > 19-hydroxyprogesterone. In conclusion, the present results show that the host-vector system used is suitable for high-level functional expression of P-45017alpha and further application of enzymatic properties of this protein to perform specific steroid biotransformations.

[The covalent-sorption reconstitution of steroid hydroxylases]. / Kovalentno-sorbtsionnaia rekonstruktsiia steroidnykh gidroksilaz.

The effect of covalent immobilization via free amino groups on the catalytic activity of individual components of the cholesterol side-chain cleavage and 11b-steroid hydroxylation systems (adrenodoxin reductase, adrenodoxin, cytochrome P-450scc and cytochrome P-450(11)b) as well as on that of co-immobilized protein complexes. The protein complex formation at different stages of the monooxygenase cycle (i.e., reduction, oxygenation) was followed by direct spectrophotometric monitoring of the functional state of the immobilized complexes. Cholesterol side-chain cleavage was carried out in minicolumns, using various combinations of immobilized and soluble proteins. Cytochromes P-450scc and P-450(11)b were found to retain their functional activities after immobilization via free SH-groups.

[Isolation and characteristics of coumarin-specific cytochrome P-450 (P-450Cho) from liver microsomes of DBA/2N mice, induced with pyrazole]. / Vydelenie i kharakteristika kumarinspetsifichnogo tsitokhroma P-450 (P-450Coh) iz mikrosom pecheni myshei DBA/2N, indutsirovannykh pirazolom.

Coumarin-specific cytochrome P-450 (P-450Coh) has been isolated from liver microsomes of DBA/2N mice induced with pyrazole. The induction effect was accompanied by a 5.8-5.9-fold increase in the P-450Coh content which made up to 14.4-17% of the total cytochrome P-450 pool in the microsomes. At the final step of P-450Coh purification, variously substituted Sepharoses (hydroxyphenyl-, cholate-, aminooctyl- and t-cytochrome-b5-) were used. The optimal scheme involved solubilization of microsomes with sodium cholate, hydrophobic chromatography on octyl-Sepharose, adsorption on calcium-tartrate gel and hydrophobic ion-exchange chromatography on aminooctyl-Sepharose. According to SDS gel electrophoresis data, the purity of P-450Coh was 95% and Mr was 50,000 Da. The amino acid composition of the protein includes 445 residues. At saturating concentrations of coumarin, more than 90% of P-450Coh are represented by the high spin form. The catalytic activity of P-450Coh was studied in reactions of xenobiotics oxidation.

Quantitation of interaction between cytochrome P-450scc and adrenodoxin--analysis in the median UV-region by second derivative spectroscopy.

Interaction between the essential protein components of the bovine adrenal mitochondrial enzyme system (cytochrome P-450scc, adrenodoxin and adrenodoxin reductase) were studied in the median UV-region utilizing second derivative difference spectroscopy. Complex formation of cytochrome P-450scc with adrenodoxin induces a signal in the second derivative difference spectrum which can be attributed to tyrosine due to its minimum at 283 nm. Based on this signal cytochrome P-450scc was titrated with adrenodoxin in dependence on different effectors (reductase, phospholipid, cholesterol). The dissociation constants (Kd) of the P-450scc/adrenodoxin complexes derived therefrom revealed an increasing affinity between both components starting from titrations in buffer solution without additional components up to the completely reconstituted system. A high affinity between P-450scc and adrenodoxin corresponds to a high turnover rate of cholesterol. Dissociation constants of the P-450scc/adrenodoxin complex were also derived from spectral changes in the Soret region. But these data do not correlate with the substrate turnover.

Purification and characterization of a liver microsomal cytochrome P-450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole-treated D2 mice.

Microsomal coumarin 7-hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612-614]. Recently we found that in D2 mice this activity is strongly and selectivity induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J. Biochem. 152-3-8]. This paper describes the purification of the pyrazole-inducible cytochrome P-450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion-exchange chromatography and the presence of 100 microM coumarin in the preparations throughout the whole purification. Coumarin effectively protected the P-450 from degradation and also converted the pyrazole-inducible P-450 to its high-spin state. This enabled us to choose only those fractions for further purification where the P-450(s) was in its high-spin state (rather than measuring the content of the total P-450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.

[Comparative study of the C27-steroid-hydroxylating system from bovine liver mitochondria]. / Sravnitel'noe izuchenie C27-steroidgidroksiliruiushchei sistemy iz mitokhondrii pecheni byka.

A method for purification of C27-steroid hydroxylating cytochrome P-450 (cytochrome P-450(27)) from bovine liver mitochondria was developed. The purification procedure included enzyme extraction from submitochondrial particles with sodium cholate, ammonium sulfate fractionation and biospecific chromatography on cholate-Sepharose and adrenodoxin-Sepharose. The resulting enzyme preparation (317-fold purification, 16% yield) was not electrophoretically homogeneous but did not contain hemoprotein admixtures. The kinetic parameters of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation in a reconstituted system containing hepatoredoxin reductase, hepatoredoxin and cytochrome P-450(27) (Km = 23 microM, kcat = 0.3 s-1 at 25 degrees C) were determined. A reciprocal functional equivalency of hepatoredoxin reductase and adrenodoxin reductase as well as of hepatoredoxin and adrenodoxin in reconstituted systems of steroid 27-hydroxylation (liver) and cholesterol side chain cleavage (adrenal cortex) was established. This equivalency was thought to be due to the similarity in essential physico-chemical properties of reductase components which was especially well-pronounced in the case of hepatoredoxin and adrenodoxin. Estimation of the functional role of lysine, dicarboxylic acid and histidine residues in ferredoxin molecules by the chemical modification method revealed the similarity of the structural organization of their protein globules: the polar residues were shown to be essential for the maintenance of native conformation; dicarboxylic acid residues formed a binding domain for the interaction with electron transport proteins, whereas histidine residues seem to participate in electron transport. At the same time, cytochrome P-450(27) and cytochrome P-450 which split the side chain of cholesterol differ in their substrate specificity, immunochemical and catalytic properties.