Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.12, #2)

Bacterial TEM-Type Serine Beta-Lactamases: Structure and Analysis of Mutations by V. G. Grigorenko; M. Yu. Rubtsova; I. V. Uporov; I. V. Ishtubaev; I. P. Andreeva; D. S. Shcherbinin; A. V. Veselovsky; A. M. Egorov (87-95).
Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2000 members: it includes genetically and functionally different bacterial enzymes capable to degrade the beta-lactam antibiotics. Beta-lactamases of molecular class A with serine residue in the active center are the most common ones. In the context of studies of the mechanisms underlying of evolution of the resistance, TEM type beta-lactamases are of particular interest due to their broad polymorphism. To date, more than 200 sequences of TEM type beta-lactamases have been described and more than 60 structures of different mutant forms of these enzymes have been presented in the Protein Data Bank. We have considered here the main structural features of the enzymes of this type with particular attention to the analysis of key mutations determining drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed a BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the TEM type beta-lactamases.
Keywords: database; TEM-type beta-lactamases; antibiotic resistance; mutant forms

Pharmacological Targets for Correction of Dyslipidemias. Opportunities and Prospects of Therapeutic Use by V. A. Kudinov; T. S. Zakharova; T. I. Torkhovskaya; O. M. Ipatova; A. I. Archakov (96-113).
The review summarizes literature data on existing pharmacological agents and novel drug preparations developed for correction of dyslipidemias and molecular mechanisms of their action. Much attention is paid to analysis of experimental and clinical studies aimed at identification of new pharmacological targets for correction of dyslipidemias and characterization of approaches increasing high density lipoproteins functionality. The implementation of alternative pharmacological agents with new alternative mechanisms of action may improve the effectiveness of traditional treatment in the future.
Keywords: cholesterol; atherosclerosis; high density lipoproteins; reverse cholesterol transport; macrophages; hypolipidemic agents

Bioinformatics Analysis of Antimicrobial Resistance Genes and Prophages Colocalized in Human Gut Metagenomes by E. V. Starikova; N. A. Prianichnikov; E. Zdobnov; V. M. Govorun (114-118).
The constant increase of bacterial antibiotic-resistant strains is directly linked to a common use of antibiotics in medicine and animal breeding. It is suggested that the gut microbiota serves as a reservoir for antibiotic resistance genes that can be transferred from symbiotic bacteria to pathogenic ones, particularly due to phage transduction. In this study, using the PHASTER prophage predicting tool and CARD antibiotics resistance database we have searched for antibiotic resistance genes that are located within prophages in human gut microbiota. After analysing metagenomic assemblies of eight samples of antibiotic treated patients, lsaE, mdfA, and cpxR/cpxA genes were identified inside prophages. These genes confer resistance to antimicrobial peptides, pleuromutilin, lincomycins, streptogramins and also multidrug resistance. Three (0.46%) of 659 putative prophages predicted in the metagenomic assemblies contained antibiotics resistance genes in their sequences.
Keywords: metagenomics; antibiotic resistance; bacteriophages

Induction of Apoptotic Endonuclease EndoG with DNA-Damaging Agents Initiates Alternative Splicing of Telomerase Catalytic Subunit hTERT and Inhibition of Telomerase Activity hTERT in Human CD4+ and CD8+ Т Lymphocytes by D. D. Zhdanov; D. A. Vasina; V. S. Orlova; E. V. Orlova; D. V. Grishin; Y. A. Gladilina; M. V. Pokrovskaya; S. S. Aleksandrova; N. N. Sokolov (119-129).
Activity of the telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At the present time, the exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and to inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ Т lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ Т cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.
Keywords: EndoG; telomerase; hTERT; alternative splicing; T lymphocytes; DNA damage

Specificity of Isatin Interaction with Cytochromes P450 by P. V. Ershov; Yu. V. Mezentsev; E. O. Yablokov; L. A. Kaluzhskiy; A. V. Florinskaya; A. V. Svirid; A. A. Gilep; S. A. Usanov; A. E. Medvedev; A. S. Ivanov (130-135).
Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this study, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase systems: five isoforms of cytochromes P450, two isoforms of cytochrome b 5, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown high affinity specific binding of isatin only to cytochromes P450 (the equilibrium dissociation constant (K d) is about 10–8 M).
Keywords: cytochrome Р450; protein-ligand interactions; isatin; surface plasmon resonance; isatin binding proteins

Myeloperoxidase Exocytosis from Activated Neutrophils in the Presence of Heparin by D. V. Grigorieva; I. V. Gorudko; V. A. Kostevich; V. B. Vasilyev; S. N. Cherenkevich; O. M. Panasenko; A. V. Sokolov (136-142).
Exocytosis of myeloperoxidase (MPO) from activated neutrophils has been investigated in the presence of the anionic polysaccharide heparin. The optimal concentration of heparin (0.1 U/mL), which did not cause additional activation of cells (lack of augmentation of lysozyme exocytosis from specific and azurophilic granules), was determined. After preincubation of cells with heparin (0.1 U/mL) MPO exocytosis from neutrophils was stimulated by various activators (fMLP, PMA, plant lectins CABA and PHA-L) and was higher as compared to the effects of the activators alone. Experiments performed using MPO isolated from leukocytes have shown that heparin in the range of concentrations 0.1–50 U/mL had no effect on MPO peroxidase activity. Thus, the use of heparin at a concentration of 0.1 U/mL avoids the artifact caused by the “loss” of MPO due to its binding to neutrophils and increases the accuracy of the method of registration of degranulation of neutrophil azurophilic granules based on determination of the MPO concentration or its peroxidase activity in cell supernatants.
Keywords: myeloperoxidase; degranulation; neutrophils; heparin; elastase; lysozyme

Construction and Characterization of a Recombinant Mutant Homolog of the CheW Protein from Thermotoga petrophila RKU-1 by D. V. Grishin; D. D. Zhdanov; Ju. A. Gladilina; V. S. Pokrovsky; O. V. Podobed; M. V. Pokrovskaya; S. S. Aleksandrova; A. L. Milyushkina; M. A. Vigovskiy; N. N. Sokolov (143-150).
In this work, a recombinant chemotaxis CheW protein from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homologue (TpeCheW-mut) have been obtained. Despite the low homology with the CheW protein of the Escherichia coli bacteria, these proteins do not cause metabolic overload and are well expressed by E. coli laboratory strains. A wide range of features and parameters important for isolation of the TpeCheW-mut protein, such as stability over a wide range of temperatures and pH values, high level of expression, solubility, and the possibility of using simple low-stage purification schemes, including heat pretreatment, has been recognized. Possible directions of using this protein in the practice of scientific and applied research have been formulated and justified.
Keywords: hyperthermophilic microorganism; recombinant thermostable CheW protein; protein overexpression; biotechnology

Comparative Subpopulation Analysis of Plasma Exosomes from Cancer Patients by S. N. Tamkovich; N. V. Yunusova; A. K. Somov; S. G. Afanas’ev; G. V. Kakurina; E. S. Kolegova; E. A. Tugutova; P. P. Laktionov; I. V. Kondakova (151-155).
Pre-selection of tumor exosomes from a total pool of circulating microvesicles is an important stage required to increase sensitivity and specificity of the developing methods for diagnostics of oncological diseases. In this study, universal proteins characterized by increased expression in plasma exosomes have been identified in patients with colorectal cancer, head and neck squamous cell carcinomas, and lung cancer. The use of antibodies against major exosomal proteins will help to develop a simple and high-performance method of affinity isolation of tumor exosomes.
Keywords: exosomes; transmission electron microscopy; flow cytometry; protein spectrum; colorectal cancer; head and neck squamous cell carcinoma; lung cancer

Representation Analysis of miRNA in Urine Microvesicles and Cell-Free Urine in Prostate Diseases by I. A. Zaporozhchenko; O. E. Bryzgunova; E. A. Lekchnov; I. D. Osipov; M. M. Zaripov; Yu. B. Yurchenko; S. V. Yarmoschuk; O. A. Pashkovskaya; E. Yu. Rykova; A. A. Zheravin; P. P. Laktionov (156-163).
Urine of prostate cancer patients contains tumor-specific biopolymers, including protein- and microvesicles-associated miRNAs that can potentially be used as oncomarkers. Previously we have characterized urinary extracellular vesicles and demonstrated diagnostic potential of their miRNAs. In this study, we have performed a comparative analysis of 84 miRNA in paired samples of urine microvesicles and urine supernatant from healthy men, patients with benign prostate hyperplasia, and prostate cancer patients using miRCURY LNA miRNA qPCR Panels. In all groups of patients, miRNA subsets characterized by different distribution between the urinary fractions have been found. In this context, two groups of miRNAs have been identified, which are involved in several signaling pathways including those associated with prostate cancer development.
Keywords: miRNA; exosomes; microvesicles; urine; prostate cancer

The protein C activator activity, determined in normal plasma by using A. ochraceus protease is comparable with the activity of a commercial protease analogue from the South American copperhead venom (Protac®). As in the case of Protac®, the A. ochraceus protease can be used for protein C determination in plasma with its reduced content. Comparison of the activator protein C activity of A. ochraceus protease and the commercial analogue showed some excess of the activator activity of the fungal preparation, which may be a promising substitute for the snake activator in diagnostical kits for determining the protein C content in clinical laboratories.
Keywords: proteases of micromycetes; activators of protein C; diagnosis of protein C

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods by T. A. Shtam; R. B. Samsonov; A. V. Volnitskiy; R. A. Kamyshinsky; N. A. Verlov; M. S. Kniazeva; E. A. Korobkina; A. S. Orehov; A. L. Vasiliev; A. L. Konevega; A. V. Malek (167-175).
Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional EV features are usually studied in vitro. Several methods of EV isolation from cell culture medium are currently used; however, selection of a particular method may have a significant impact on obtained results. The choice of the optimal method is usually determined by the amount of starting biomaterial and the aims of the research. We have performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with 30% sucrose/D2O “cushion,” precipitation with plant proteins and latex-based immunoaffinity capturing. EV isolated from several human glial cell lines by different approaches were compared in terms of the following parameters: size, concentration, EV morphology, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, total RNA, and several glioma-associated miRNAs. The applied methods included nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. Based on the obtained results, we have developed practical recommendations that may help researchers to make the best choice of the EV isolation method.
Keywords: extracellular vesicles; exsosomes; methods of isolation; ultracentrifugation; lectines; immunoprecipitation

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring by K. G. Ptitsyn; S. E. Novikova; Y. Y. Kiseleva; A. A. Moysa; L. K. Kurbatov; T. E. Farafonova; S. P. Radko; V. G. Zgoda; A. I. Archakov (176-180).
The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of the target protein with aptamers immobilized on a solid phase has been investigated. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as a model target protein. The affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads resulted in an approximately 10-fold increase of the concentration of the target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM was possible without interference from other peptides present in the tryptic digest.
Keywords: aptamers; thrombin; enrichment; quantitative detection; selected reaction monitoring

Organophosphorus pesticides (OP) are used to protect crops from pests. Treatment of plants and animals with pesticides can be done during their growth or creation of conditions required for the long-shelf life of the agricultural products. Currently, many remedies exist for prevention and removal of intoxication consequences developed in living organisms exposed to OPs. The development of biologics for degradation of OPs and biotechnologies for their application in agriculture still represents an important task. New biologics based on stabilized forms of such enzyme as hexahistidine-tagged organophosphorus hydrolase (His6-OPH) have been in the form of nano-sized particles for OPs detoxification. They represent enzyme-polyelectrolyte complexes (EPC) obtained by mixing solutions of His6-OPH and polyanion under certain conditions. The main purpose of this work was to evaluate the usage efficiency of EPC based on His6-OPH and polyglutamic acid for OPs detoxification by analyzing biochemical blood parameters of rats fed with a grain mix containing chlorpyrifos. The experiment was conducted using female Sprague-Dowly albino rats. Treatment of the feeding grain mix initially containing chlopyrifos (48 mg/kg of the mix) with EPC based on His6-OPH (1000 U/kg of the mix) for 24 h was the most effective. The results showed that acetyl cholinesterase activity in blood of rats from the group consuming food after the enzymatic removal of chlorpyrifos, was comparable to acetyl cholinesterase activity in blood of rats consuming pure food.
Keywords: hexahistidine-tagged organophosphorus hydrolase; organophosphorus pesticides; hydrolysis; biochemical blood analysis

Rats with experimental parkinsonism induced by intraperitoneal daily administration of rotenone (2.75 mg/kg) during seven days are characterized by an increased activity of prolyl endopeptidase (PEP; ЕС 3.4.21.26) in serum and a decreased activity of adenosine deaminase (ADA; EC 3.5.4.4) in serum and in the prefrontal cortex. PEP and ADA activities in other brain structures (in the striatum, hypothalamus, and hippocampus) as well as activity of dipeptidyl peptidase IV (DPP-4, CD26; ЕС 3.4.14.5) in serum and in all the brain structures investigated remained unchanged. Afobazole and levodopa, which exhibit antiparkinsonian activity in this model of parkinsonism, decreased the elevated PEP activity in serum and increased ADA activity reduced in the prefrontal cortex of rats with the experimental pathology. At the same time, treatment with these drugs resulted in a decrease of ADA activity in the other brain structures.
Keywords: parkinsonism; dipeptidyl peptidase IV; prolyl endopeptidase; adenosine deaminase; rotenone; rats