Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.12, #3)

The Role of Exosomal Tetraspanins and Proteases in Tumor Progression by N. V. Yunusova; E. A. Tugutova; S. N. Tamkovich; I. V. Kondakova (191-202).
Major (CD9, CD63, CD81) and other (CD82, CD151, Tspan8) exosomal tetraspanins interact with various proteins and form functional tetraspanin complexes in exosomes. The tetraspanin complexes contain various proteins including proteases. Tetraspanin-associated exosomal proteases (ADAM proteases, MMPs, EMMPRIN) play an important role in cell motility, migration, invasion and formation of metastases. Proteases that are not associated with tetraspanins also significantly contribute to tumor progression. They destabilize intercellular contacts, promote migration and invasion of tumor cells, and participate in regulation of the expression IGF-I, VEGF, and the activated forms of transcription factors. The role of other exosomal proteases in tumor progression is being clarified.
Keywords: exosomes; tetraspanins; proteases; tumor progression

Ubiquitin-Independent Degradation of Proteins in Proteasomes by O. A. Buneeva; A. E. Medvedev (203-219).
Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular proteins. Until recently, it was generally accepted that prior to proteolytic degradation in proteasomes the proteins had to be targeted by ubiquitination: ATP-dependent attachment of (typically four sequential) residues of the low-molecular protein, ubiquitin, which involves the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase. Cytoplasmic and nucleoplasmic proteins labeled in this way are then digested in 26S proteasomes. However, it becomes increasingly clear that using this route the cell eliminates only a part of unwanted proteins. Many proteins can be cleaved by the 20S proteasome in an ATP-independent manner and without previous ubiquitination. Ubiquitin-independent degradation of proteins in proteasomes is a relatively new area of studies of the role of the ubiquitin-proteasome system. However, recent data obtained in this direction already correct existing concepts about proteasomal degradation of proteins and its regulation. Ubiquitin-independent proteasome degradation needs the main structural precondition in proteins: the presence of unstructured regions in the amino acid sequences that provide interaction with the proteasome. Taking into consideration that in humans almost half of all genes encode proteins that contain a certain proportion of intrinsically disordered regions, it appears that the list of proteins undergoing ubiquitin-independent degradation will demonstrate a further increase. Since 26S proteasomes account for only 30% of the total proteasome content in mammalian cells, most of the proteasomes exist in the form of 20S complexes. The latter suggests that ubiquitin-independent proteolysis performed by the 20S proteasome is a natural process of removing damaged proteins from the cell and maintaining a constant level of intrinsically disordered proteins. In this case, the functional overload of proteasomes in aging and/or other types of pathological processes, if it is not accompanied by triggering more radical mechanisms for the elimination of damaged proteins, organelles, and whole cells, has the most serious consequences for the whole organism.
Keywords: ubiquitin-proteasome protein degradation system; proteasome; ubiquitin-independent protein degradation

Methods for Determination of Functional Activity of Cytochrome P450 Isoenzymes by A. V. Kuzikov; R. A. Masamrekh; A. I. Archakov; V. V. Shumyantseva (220-240).
The review deals with modern methods and technologies for determination of functional activity cytochrome P450 isoenzymes; these include absorbance and fluorescent spectroscopy, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), Raman, Mössbauer, and X-ray spectroscopy, surface plasmon resonance (SPR), atomic force microscopy (AFM). Methods of molecular genetic analysis have been considered in the context of personalized medicine. Using the methods of chromatography mass spectrometry it is possible to analyze reaction products formed in reactions catalyzed by cytochromes P450. Special attention is paid to modern electrochemical systems based on cytochrome P450 isoenzymes, their applicability for analysis of their catalytic activity, their use in practice and further development perspectives for experimental pharmacology, biotechnology and translational medicine.
Keywords: cytochrome P450; substrates; inhibitors; catalysis; electrochemical analysis; spectroscopy; mass spectrometry

The Analysis of Participation of Individual Proteins in the Protein Interactome Formation by A. V. Florinskaya; P. V. Ershov; Yu. V. Mezentsev; L. A. Kaluzhskiy; E. O. Yablokov; O. A. Buneeva; V. G. Zgoda; A. E. Medvedev; A. S. Ivanov (241-246).
It becomes increasingly clear that most proteins of living systems exist as components of various protein complexes rather than individual molecules. The use of various proteomic techniques significantly extended our knowledge not only about functioning of individual complexes but also formed a basis for systemic analysis of protein-protein interactions. In this study gel-filtration chromatography accompanied by mass spectrometry was used for the interactome analysis of human liver proteins. In six fractions (with average molecular masses of 45 kDa, 60 kDa, 85 kDa, 150 kDa, 250 kDa, and 440 kDa) 797 proteins were identified. In dependence of their distribution profiles in the fractions, these proteins could be subdivided into four groups: (1) single monomeric proteins that are not involved in formation of stable protein complexes; (2) proteins existing as homodimers or heterodimers with comparable partners; (3) proteins that are partially exist as monomers and partially as components of protein complexes; (4) proteins that do not exist in the monomolecular state, but also exist within protein complexes containing three or more subunits. Application of this approach to known isatin-binding proteins resulted in identification of proteins involved in formation of the homo- and heterodimers and mixed protein complexes.
Keywords: protein-protein interactions; protein interactomics; chromatographic fractionation of biological material; protein mass spectrometry; systems analysis

Phospholipase D: Its Role in Metabolic Processes and Development of Diseases by G. V. Ramenskaia; E. V. Melnik; A. E. Petukhov (247-257).
Phospholipase D (PLD; EC 3.1.4.4) is one of the key enzymes catalyzing hydrolysis of cell membrane phospholipids. This review considers and summaries current knowledge about six human PLD isoforms, their structure and a role in physiological and pathological processes. Comparative analysis of PLD isoforms structure is presented. The review considers the mechanism of hydrolysis and transphosphatidylation performed by PLD, the role of PLD1 and PLD2 in the pathogenesis of some types of cancer, infectious, thrombotic, and neurodegenerative diseases is analyzed. The prospects of development of PLD isoformselective inhibitors are considered in the context of their clinical use and inclusion into various therapeutic schemes; the latter is especially important in the case of already developed PLD inhibitors. Phosphatidylethanol (PEth) formed in the human body during phospholipid transphosphatidylation catalyzed by PLD is considered as an alcohol abuse biomarker.
Keywords: phospholipase D (PLD); phospholipase D isoforms; phospholipase D inhibitors; phosphatidylethanol (PEth); alcohol abuse biomarkers

Enzymatic and Bactericidal Activity of Monomeric and Dimeric Forms of Myeloperoxidase by T. V. Vakhrusheva; A. V. Sokolov; V. A. Kostevich; V. B. Vasilyev; O. M. Panasenko (258-265).
Enzymatic and bactericidal activities of mature, dimeric myeloperoxidase (MPO) and its monomeric form have been compared. Dimeric MPO was isolated from HL-60 cells. Hemi-MPO obtained from dimeric MPO by reductive cleavage of a disulfide bond between protomeric subunits was used as the monomeric form. Both peroxidase and halogenating (chlorinating) activities of MPO were assayed, each by two methods. Bactericidal activity of the MPO/Н2О2/Cl‾ system was tested using the Escherichia coli laboratory strain DH5α. No difference in the enzymatic and bactericidal activity between dimeric MPO and hemi- MPO was found. Both forms of the enzyme also did not differ in the resistance to HOCl, the main product of MPO. HOCl caused a dose-dependent decrease in peroxidase and chlorinating activity, and the pattern of this decrease was identical for dimeric MPO and hemi-MPO. At the equal heme concentration, the hemi- MPO/Н2О2/Cl‾ system demonstrated a somewhat higher bactericidal effect than the dimeric MPO/Н2О2/Cl‾ system. This is most likely explained by higher probability of contacts between the bacterial surface and hemi-MPO molecules, since at the same heme concentration the number of hemi-MPO molecules is 2-fold higher than that of dimeric MPO molecules. Using Western-blotting with antibodies to MPO, we have shown, for the first time, that the dimeric molecule of MPO could be cleaved into two monomeric subunits by HOCl, most probably due to oxidation of the disulfide bond between these subunits. This suggests that appearance in blood of MPO with mass corresponding to its monomer may result from the damage of dimeric MPO by reactive halogen species, especially upon their overproduction inducing oxidative/halogenative stress in inflammatory diseases.
Keywords: myeloperoxidase; hemi-myeloperoxidase; chlorinating activity; peroxidase activity; bactericidal myeloperoxidase system; oxidative/halogenative stress

The Role of Single Nucleotide Polymorphisms in the GIPR Gene in Regulation of Secretion of Hormones and Adipokines in Obese Patients with Type 2 Diabetes Mellitus by D. A. Skuratovskaia; M. A. Vulf; E. V. Kirienkova; N. I. Mironyuk; P. A. Zatolokin; L. S. Litvinova (266-274).
The relationship between the rs2302382, rs8111428 and Glu354Gln (rs1800437) polymorphisms in the GIPR (glucose-dependent insulinotropic polypeptide receptor) gene and plasma levels of mediators involved in the regulation of carbohydrate metabolism has been investigated in obese patients with type 2 diabetes mellitus (T2DM) before and after test breakfast. The contribution of the polymorphic variants of rs2302382, rs8111428 in the GIPR gene contributed to T2DM genetic predisposition in Russian individuals belonging to the Slavic population. Polymorphisms rs2302382 and rs8111428 in the GIPR gene were characterized by the linkage disequilibrium. The decrease in the expression level of the GIPR gene in mesenteric adipose tissue of the small intestine in the carriers of the CC genotype rs2302382 and AA rs8111428 was associated with the increase in the plasma leptin level, whereas during its normal expression, the plasma content of insulin, and GIP (in persons with the CA genotype of the polymorphism rs2302382 and AG polymorphism rs8111428), resistin and ghrelin (in individuals with the CA genotype of the polymorphism rs2302382) increased. We propose the stimulating effect of GIP on the secretion of resistin, leptin and ghrelin, with an increase in insulin production in obese patients with T2DM.
Keywords: incretins; adipokines; glucose-dependent insulinotropic polypeptide; hormones; gene expression; polymorphism

Primary Screening of Photosensitizers of the Bacteriochlorin Series for Photodynamic Therapy of Malignant Neoplasms by E. A. Plotnikova; M. A. Grin; P. V. Ostroverkhov; I. V. Pantushenko; R. I. Yakubovskaya; A. D. Kaprin (275-282).
Primary screening of photosensitizers of the bacteriochlorin series containing aminoamide, propyl, and carbohydrate substituents was carried out; it included an assessment of spectral characteristics of these compounds, their storage stability in solution under darkened conditions and light exposure, and the study of photoinduced activity and cytotoxicity on tumor of HEp2 cells. According to the results of absorption and fluorescence analyzes, dyes with aminoamide substituents had a maximum at 754 ± 2 nm, are not stable during storage (fluorescence intensity decreased by 33–56% during a day); compounds with propyl and carbohydrate substituents absorbed in the region of 780–831 nm were characterized by stability during storage and irradiation. All PS except the dye with the carbohydrate residue in the exocycle E, exhibited photoinduced activity and lack of dark toxicity. The highest photoactivity was detected in compounds with aminoamide fragments in the macrocyclic ring (during 2 h-incubation the IC50 ranged from 17 nM to 49 nM). Taking into consideration physicochemical and biological properties, as well as technology of production and stability during storage and irradiation, O-propyloxime-N-propoxybacteriopurinimide methyl ester was chosen for further research aimed at creating a new PS generation for photodynamic therapy of malignant tumors.
Keywords: bacteriochlorophyll а ; bacteriochlorin; photodynamic therapy; photosensitizers; photo-induced activity in vitro; primary screening