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1.
Biomed Khim ; 69(5): 253-280, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37937429

ABSTRACT

Traditional antiviral vaccines are currently created by inactivating the virus chemically, most often using formaldehyde or ß-propiolactone. These approaches are not optimal since they negatively affect the safety of the antigenic determinants of the inactivated particles and require additional purification stages. The most promising platforms for creating vaccines are based on pseudoviruses, i.e., viruses that have completely preserved the outer shell (capsid), while losing the ability to reproduce owing to the destruction of the genome. The irradiation of viruses with electron beam is the optimal way to create pseudoviral particles. In this review, with the example of the poliovirus, the main algorithms that can be applied to characterize pseudoviral particles functionally and structurally in the process of creating a vaccine preparation are presented. These algorithms are, namely, the analysis of the degree of genome destruction and coimmunogenicity. The structure of the poliovirus and methods of its inactivation are considered. Methods for assessing residual infectivity and immunogenicity are proposed for the functional characterization of pseudoviruses. Genome integrity analysis approaches, atomic force and electron microscopy, surface plasmon resonance, and bioelectrochemical methods are crucial to structural characterization of the pseudovirus particles.


Subject(s)
Poliomyelitis , Poliovirus , Vaccines , Humans , Poliovirus/genetics , Formaldehyde , Propiolactone , Poliomyelitis/prevention & control
2.
Biomed Khim ; 68(1): 18-31, 2022 Jan.
Article in Russian | MEDLINE | ID: mdl-35221293

ABSTRACT

We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.


Subject(s)
Proteasome Endopeptidase Complex , Ubiquitinated Proteins , Animals , Brain/metabolism , Liver/metabolism , Proteasome Endopeptidase Complex/metabolism , Rabbits , Ubiquitin/metabolism
3.
Biomed Khim ; 68(1): 47-54, 2022 Jan.
Article in Russian | MEDLINE | ID: mdl-35221296

ABSTRACT

The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻6 M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻8 M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.


Subject(s)
Adrenodoxin , Cholesterol Side-Chain Cleavage Enzyme , Adrenodoxin/chemistry , Adrenodoxin/genetics , Adrenodoxin/metabolism , Kinetics , Mitochondria/metabolism , Oxidation-Reduction
4.
Biomed Khim ; 67(1): 42-50, 2021 Jan.
Article in Russian | MEDLINE | ID: mdl-33645521

ABSTRACT

Currently, opportunistic fungi of the genus Candida are the main causative agents of mycoses, which are especially severe upon condition of acquired immunodeficiency. The main target for the development of new antimycotics is the cytochrome P450 51 (CYP51) of the pathogenic fungus. Due to the widespread distribution of Candida strains resistancy to inhibitors of the azole class, the screening for CYP51 inhibitors both among non-azole compounds and among clinically used drugs repurposing as antimycotics is becoming urgent. To identify potential inhibitors from the non-azole group, an integrated approach was applied, including bioinformatics analysis, computer molecular modeling, and a surface plasmon resonance (SPR) technology. Using in silico modeling, the binding sites for acetylsalicylic acid, ibuprofen, chlorpromazine and haloperidol (this compounds, according to the literature, showed antimycotic activity) were predicted in the active site of CYP51 of Candida albicans and Candida glabrata. The Kd values of molecular complexes of acetylsalicylic acid, ibuprofen and haloperidol with CYP51, determined by SPR analysis, ranged from 18 µM to 126 µM. It was also shown that structural derivatives of haloperidol, containing various substituents, could be positioned in the active site of CYP51 of Candida albicans with the possible formation of coordination bonds between the hydroxyl groups of the derivatives and the iron atom in the heme of CYP51. Thus, the potential basic structures of non-azole compounds have been proposed, which can be used for the design of new CYP51 inhibitors of Candida fungi.


Subject(s)
Antifungal Agents , Candida , 14-alpha Demethylase Inhibitors/pharmacology , Antifungal Agents/pharmacology , Candida albicans , Cytochrome P-450 Enzyme System , Sterol 14-Demethylase
5.
Biomed Khim ; 67(1): 51-65, 2021 Jan.
Article in Russian | MEDLINE | ID: mdl-33645522

ABSTRACT

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.


Subject(s)
Isatin , Neurotoxins , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Animals , Brain/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Isatin/metabolism , Isatin/pharmacology , Mice , Mitochondria/metabolism , Neurotoxins/metabolism , Neurotoxins/pharmacology , Proteomics
6.
Biomed Khim ; 65(4): 306-310, 2019 Jun.
Article in Russian | MEDLINE | ID: mdl-31436171

ABSTRACT

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.


Subject(s)
Biosensing Techniques , Proteasome Endopeptidase Complex/chemistry , Ubiquitinated Proteins/chemistry , Humans , Ubiquitin , Ubiquitination
7.
Biomed Khim ; 65(1): 63-66, 2019 Jan.
Article in Russian | MEDLINE | ID: mdl-30816099

ABSTRACT

Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Intramolecular Oxidoreductases/metabolism , Humans , Microsomes/enzymology , Mitochondria/enzymology , Prostaglandins I/biosynthesis , Thromboxane A2/biosynthesis
8.
Biomed Khim ; 64(5): 423-428, 2018 Sep.
Article in Russian | MEDLINE | ID: mdl-30378558

ABSTRACT

Amyloid-ß peptide (1-42) (Aß1-42) is a key player in the development and progression of Alzheimer's disease (AD) and related pathologies, determined by formation of protein aggregates in the central nervous system. Aß1-42 binding to crucial intracellular targets (and their subsequent inactivation) obviously represents one of the earliest events preceding extracellular pathogenic oligomerization/aggregation of Aß1-42. It is reasonable to expect that dissociation of the Aß1-42 complexes with intracellular proteins by means of inhibitors followed by subsequent degradation of Aß1-42 would not only protect critically important proteins but also prevent intracellular accumulation of Aß1-42. The aim of this study was to investigate the effect of the neuroprotector isatin (100 mM) on interaction of known Aß-binding proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase, with Aß1-42 and its fragments (Aß1-28, Aß12-28, Aß25-35). Aß1-42 and its fragments (Aß1-28, Aß12-28, Aß25-35) immobilized on the Biacore optical biosensor chip interacted with GAPDH and pyruvate kinase. The lowest and basically equal Kd values were determined for GAPDH and pyruvate kinase complexes with immobilized Aß1-42 and Aß25-35. The presence of 100 mM isatin caused a significant (more than fivefold) increase in the Kd values for GAPDH complexes with all Aß peptides except Aß1-28. In contrast to GAPDH isatin increased dissociation of pyruvate kinase complexes only with Aß1-42 (causing a 30-fold increase in Kd) and to a lesser extent with Aß12-28 and Aß25-35 (a 10-fold increase in Kd). It should be noted that in the presence of isatin the Kd values for GAPDH and pyruvate kinase complexes with all Aß studied were in a narrower concentration range (10-7 M - 10-6 M) than in the absence of this neuroprotector (10-8 M - 10-6 M). Data obtained suggest existence of principal possibility of (pharmacological) protection of crucial intracellular targets against both Aß1-42, and its aggressive truncated peptides (Aß25-35).


Subject(s)
Isatin/metabolism , Alzheimer Disease , Amyloid beta-Peptides , Glyceraldehyde-3-Phosphate Dehydrogenases , Humans , Peptide Fragments
9.
Biochemistry (Mosc) ; 82(9): 1042-1047, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28988533

ABSTRACT

Recent proteomic profiling of mouse brain preparations using the ubiquitin receptor, Rpn10 proteasome subunit, as an affinity ligand revealed a representative group of proteins bound to this sorbent (Medvedev, A. E., et al. (2017) Biochemistry (Moscow), 82, 330-339). In the present study, we investigated interaction of the Rpn10 subunit of proteasomes with some of these identified proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and histones H2A and H2B. The study revealed: (i) quantitative affinity interaction of the proteasome subunit immobilized on a Biacore-3000 optical biosensor cuvette with both the GAPDH (Kd = 2.4·10-6 M) and pyruvate kinase (Kd = 2.8·10-5 M); (ii) quantitative high-affinity interaction of immobilized histones H2A and H2B with the Rpn10 subunit (Kd values of 6.5·10-8 and 3.2·10-9 M, respectively). Mass spectrometric analysis revealed the presence of the ubiquitin signature (GG) only in a highly purified preparation of GAPDH. We suggest that binding (especially high-affinity binding) of non-ubiquitinated proteins to the Rpn10 proteasome subunit can both regulate the functioning of this proteasomal ubiquitin receptor (by competing with ubiquitinated substrates) and promote activation of other pathways for proteolytic degradation of proteins destined to the proteasome.


Subject(s)
Proteasome Endopeptidase Complex/metabolism , Ubiquitinated Proteins/metabolism , Animals , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Histones/metabolism , Humans , Kinetics , Protein Binding , Pyruvate Kinase/metabolism , RNA-Binding Proteins , Rabbits
10.
Biomed Khim ; 63(2): 170-175, 2017 Mar.
Article in Russian | MEDLINE | ID: mdl-28414290

ABSTRACT

Cytochromes P450 (CYP) are involved in numerous biochemical processes including metabolism of xenobiotics, biosynthesis of cholesterol, steroid hormones etc. Since some CYP catalyze indol oxidation to isatin, we have hypothesized that isatin can regulate protein-protein interactions (PPI) between components of the CYP system thus representing a (negative?) feedback mechanism. The aim of this study was to investigate a possible effect of isatin on interaction of human CYP with cytochrome b5 (CYB5A). Using the optical biosensor test system employing surface plasmon resonance (SPR) we have investigated interaction of immobilized CYB5A with various CYP in the absence and in the presence of isatin. The SPR-based experiments have shown that a high concentration of isatin (270 mM) increases Kd values for complexes CYB5A/CYP3А5 and CYB5A/CYP3A4 (twofold and threefold, respectively), but has no influence on complex formation between CYB5A and other CYP (including indol-metabolizing CYP2C19 and CYP2E1). Isatin injection to the optical biosensor chip with the preformed molecular complex CYB5A/CYP3A4 caused a 30%-increase in its dissociation rate. Molecular docking manipulations have shown that isatin can influence interaction of CYP3А5 or CYP3A4 with CYB5A acting at the contact region of CYB5A/CYP.


Subject(s)
Cytochrome P-450 CYP2C19/chemistry , Cytochrome P-450 CYP2E1/chemistry , Cytochrome P-450 CYP3A/chemistry , Cytochromes b5/chemistry , Isatin/chemistry , Binding Sites , Cholesterol Side-Chain Cleavage Enzyme/chemistry , Cytochrome P-450 CYP2C9/chemistry , Humans , Kinetics , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Solutions , Steroid 11-beta-Hydroxylase/chemistry , Steroid 17-alpha-Hydroxylase/chemistry , Surface Plasmon Resonance
11.
Biomed Khim ; 62(6): 720-724, 2016 Nov.
Article in Russian | MEDLINE | ID: mdl-28026818

ABSTRACT

The amyloid-beta peptide 1-42 formed during proteolytic processing of the amyloid precursor protein (APP) plays a key role in the development or progression of Alzheimer's disease (AD) and other pathologies associated with formation of protein aggregates in the central nervous system. Recent proteomic profiling of mouse and rat brain preparations by means of beta-amyloid peptide immobilized on Affigel-10 revealed a large group of amyloid-binding proteins (n>80). Many (about 25%) of these proteins were previously identified as isatin-binding proteins. The aim of this study was to validate direct interaction between beta-amyloid peptide and highly purified intact and oxidized peroxiredoxin, M-type pyruvate kinase, alpha-enolase, and the effect of isatin on this interaction. The study performed using SPR-based Biacore 3000 and Biacore X100 biosensors has shown that all the proteins form molecular complexes with immobilized beta-amyloid peptide. The Kd values for these complexes varied from 8.36х10-8 M (peroxiredoxin) to 1.97х10-6 M (alpha-enolase). Oxidative modification of investigated proteins caused opposite effects on complexes of these peptides with beta-amyloid. The endogenous neuroprotector isatin increased dissociation of complexes formed by beta-amyloid peptide with both intact proteins (peroxiredoxin, glyceraldehyde-3-phosphate dehydrogenase) and/or oxidized proteins (peroxiredoxin, pyruvate kinase) used in this study.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Isatin/chemistry , Neuroprotective Agents/chemistry , Surface Plasmon Resonance , Amyloid beta-Protein Precursor/metabolism , Animals , Humans , Isatin/metabolism , Mice , Neuroprotective Agents/metabolism , Protein Binding , Rats
12.
Biomed Khim ; 62(2): 160-3, 2016.
Article in Russian | MEDLINE | ID: mdl-27143373

ABSTRACT

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classical glycolytic redox sensitive enzyme, exhibits various non-glycolytic functions, which are considered to be especially important for progression of various neurodegenerative diseases. GAPDH binds isatin (indole-dione-2,3), an endogenous indole often used as a parent component in numerous derivatives demonstrating diverse pharmacological (including neuroprotector) activities. In this study we have investigated binding of intact and mildly oxidized GAPDH to immobilized isatin, using an optical biosensor technique, employing surface plasmon resonance (SPR), and the effect of isatin as a probe for this binding. Mild GAPDH oxidation by 70 mM H(2)O(2) increased enzyme dissociation from immobilized isatin. Since GAPDH is considered as a putative target for various neuroprotector agents, this suggests that its redox state determines sensitivity to neuroprotective agents, and oxidative stress typical for various neurodegenerative disorders may significantly reduce pharmacological effectiveness of such compounds.


Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Isatin/metabolism , Biosensing Techniques , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Isatin/chemistry , Neuroprotective Agents/pharmacology , Oxidation-Reduction , Oxidative Stress , Surface Plasmon Resonance
13.
Biomed Khim ; 61(5): 632-5, 2015.
Article in Russian | MEDLINE | ID: mdl-26539872

ABSTRACT

Isatin-binding activity of mice liver proteins has been investigated in the samples from the control and flight groups by using the methods of biosensor and proteomic analysis. It was found the higher isatin-binding activity in mice of flight group. The content of a number of individual isatin-binding proteins in the samples of the flight groups differ slightly from the ground control. However, in samples from animals which have weekly post-flight adaptation, the level of certain proteins was significantly increased. The latter allows us to assume that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins), occurs in early post-flight period.


Subject(s)
Adaptation, Physiological , Carrier Proteins/metabolism , Isatin/chemistry , Space Flight , Actins/isolation & purification , Actins/metabolism , Alcohol Dehydrogenase/isolation & purification , Alcohol Dehydrogenase/metabolism , Aldehyde Dehydrogenase/isolation & purification , Aldehyde Dehydrogenase/metabolism , Animals , Carrier Proteins/isolation & purification , Glyceraldehyde-3-Phosphate Dehydrogenases/isolation & purification , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Liver/chemistry , Mice , Mice, Inbred C57BL , Peroxiredoxins/isolation & purification , Peroxiredoxins/metabolism , Protein Binding , Proteome/isolation & purification , Proteome/metabolism , Time Factors , Weightlessness
14.
Biomed Khim ; 61(4): 468-73, 2015.
Article in Russian | MEDLINE | ID: mdl-26350737

ABSTRACT

Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.


Subject(s)
Cytochrome P-450 CYP3A/chemistry , Cytochromes b5/chemistry , Biocatalysis , Electrochemical Techniques , Enzymes, Immobilized/chemistry , Humans , Isoenzymes/chemistry , Microsomes/chemistry , Microsomes/metabolism , Mitochondria/chemistry , Mitochondria/metabolism , Oxidation-Reduction , Protein Array Analysis , Protein Binding , Recombinant Proteins/chemistry , Surface Plasmon Resonance
15.
Biomed Khim ; 60(5): 528-37, 2014.
Article in Russian | MEDLINE | ID: mdl-25386880

ABSTRACT

The cholesterol biosynthesis regulation is the important part of the hypercholesterolemia diseases therapy. The inhibition of the post-squalene cholesterol biosynthesis steps provide the alternative to classic statin therapy. Sterol-14a-demethylase (CYP51) is one of the hypothetical targets for it. In this work the screening of the ability to interact with human CYP51 (CYP51A1) for the nature low-weight compounds with steroid-like scaffold were performed by integration of the surface plasmon resonance biosensor and spectral titration methods. The results of the selection were 4 compounds (betulafolientriol, holothurin A, teasaponin, capsicoside A) witch had high affinity to the CYP51A1 active site. These data extend the range of compounds which may be used as specific inhibitors of CYP51 and give the permission to suggest the dynamic of the enzyme.


Subject(s)
14-alpha Demethylase Inhibitors/pharmacology , Lanosterol/pharmacology , Sterol 14-Demethylase/metabolism , 14-alpha Demethylase Inhibitors/chemistry , Humans , Lanosterol/analogs & derivatives , Lanosterol/chemistry , Protein Binding , Surface Plasmon Resonance
16.
Biomed Khim ; 60(1): 17-27, 2014.
Article in Russian | MEDLINE | ID: mdl-24749245

ABSTRACT

Molecular interactions between proteins redox partners (cytochromes P450 3A4, 3A5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 - -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.


Subject(s)
Cytochrome P-450 CYP3A/chemistry , Cytochromes b5/chemistry , Microsomes/chemistry , Mitochondrial Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Biocatalysis , Biosensing Techniques , Cytochrome P-450 CYP3A/genetics , Cytochromes b5/genetics , Electrochemical Techniques , Electrodes , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/genetics , Kinetics , Mitochondrial Proteins/genetics , Oxidation-Reduction , Protein Binding , Recombinant Fusion Proteins/genetics , Solutions , Testosterone/chemistry
17.
Biomed Khim ; 60(6): 615-22, 2014.
Article in Russian | MEDLINE | ID: mdl-25552499

ABSTRACT

Protein ubiquitination is considered as an important mechanism that is responsible not only for specific labeling of proteins for their subsequent degradation but also for localization of proteins in the cell and regulation of protein-protein interactions. In the context of protein-protein interactions binding of (mono/poly)ubiquitinated molecules to proteins containing specific ubiquitin binding domains appear to play the decisive role. Although formation of the ubiquitin interactome has been demonstrated for cytosol, involvement of mitochondria and associated extramitochondrial proteins into such interactions still requires detailed investigation. In this study using an optical biosensor we have demonstrated binding of proteins of mouse brain mitochondrial lysates to immobilized monomeric ubiquitin. Model purified proteins, which are known to be associated with the outer mitochondrial compartment (glyceraldehyde-3-phosphate dehydorgenase, creatine phosphokinase), interacted with immobilized ubiquitin as well as with each other. This suggests that (poly)ubiquitinated chains may be involved in protein-protein interactions between ubiquitinated and non-ubiquitinated proteins and thus may contribute to formation of (mitochondrial) ubiquitin subinteractome.


Subject(s)
Creatine Kinase/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating)/metabolism , Immobilized Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Protein Processing, Post-Translational , Ubiquitin/metabolism , Animals , Biosensing Techniques , Brain Chemistry , Cattle , Creatine Kinase/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating)/chemistry , Immobilized Proteins/chemistry , Mice , Mitochondria/chemistry , Mitochondrial Proteins/chemistry , Muscle, Skeletal/chemistry , Protein Binding , Rabbits , Ubiquitin/chemistry , Ubiquitination
18.
Biomed Khim ; 59(4): 388-98, 2013.
Article in Russian | MEDLINE | ID: mdl-24502137

ABSTRACT

The SPR assay for human cytochrome P450 51A1's (CYP51A1) ligand screening was developed. Assay has been validated with known azole inhibitors of cytochrome P450s. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR assay was showed with 19 steroid and triterpene compounds, which were not investigated as potential ligands of CYP51A1.


Subject(s)
14-alpha Demethylase Inhibitors/chemistry , Biosensing Techniques , Ketoconazole/chemistry , Sterol 14-Demethylase/chemistry , 14-alpha Demethylase Inhibitors/metabolism , Humans , Ketoconazole/metabolism , Protein Binding , Sterol 14-Demethylase/metabolism
19.
Biomed Khim ; 58(1): 43-9, 2012.
Article in Russian | MEDLINE | ID: mdl-22642151

ABSTRACT

Here, we describe the analysis of kinetic and thermodynamic parameters for binding of peptide and nonpeptide dimerization inhibitors to immobilized HIV protease (HIVp) monomers by using surface plasmon resonance. Molecular interactions were investigated at different inhibitors concentrations (0-80 microM) and temperatures (15-35 degrees C). The kinetic, equilibrium and thermodynamic parameters have been determined. It was found that both inhibitors were characterized by similar interaction parameters. The complex formation is entropically driven process for both inhibitors. The entropic term(-TdeltaS) had the value about -20 kcal/mol while the enthalpic term (deltaH) had the positive value about 14 kcal/mol and counteracted the complex formation.


Subject(s)
HIV Protease Inhibitors/chemistry , HIV Protease/chemistry , Protein Multimerization/drug effects , Surface Plasmon Resonance/methods , Dimerization , Humans , Kinetics , Protein Binding , Thermodynamics
20.
Biomed Khim ; 56(3): 405-12, 2010.
Article in Russian | MEDLINE | ID: mdl-20695220

ABSTRACT

Aptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide constructs with use of a poly-(dT)-linker of 35 nucleotides (nt) long. Complexes of thrombin with the aptamers and their hetero- and homodimeric constructs were measured using the optical biosensor Biacore-3000. K(D) values measured for the hetero- and homodimeric constructs were correspondingly 25-30- and 2-3-fold lower than those for the primary aptamers. Analysis of temperature dependencies of K(D) values within the temperature interval of 10 degrees C-40 degrees C has shown that the values of enthalpy change deltaH upon formation of complexes of thrombin with the aptamers and the hetrodimeric construct are close. The value of the entropy change deltaS upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5-2-fold higher than deltaS values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of temperature from 10 degrees C to 37 degrees C. However, the dissociation rate for the complex of thrombin with the heterodimeric construct was evidently lower that that for the complexes with the aptamers.


Subject(s)
Aptamers, Nucleotide/chemistry , Thrombin/chemistry , Biosensing Techniques , Dimerization , Temperature , Thermodynamics , Thrombin/antagonists & inhibitors
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