Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.11, #2)

Furin as proprotein convertase and its role in normal and pathological biological processes by N. I. Solovyeva; T. A. Gureeva; O. S. Timoshenko; T. A. Moskvitina; E. V. Kugaevskaya (87-100).
Furin belongs to intracellular serine Ca2+-dependent endopeptidases of the subtilisin family, also known as proprotein convertases (PC). Human furin is synthesized as a zymogen with a molecular weight of 104 kDа, which is then autocatalytically activated in two stages. This process occurs during zymogen migration from the endoplasmic reticulum to the Golgi apparatus, where a large part of furin is accumulated. The molecular weight of the active furin is 98 kDа. Furin is the enzyme with narrow substrate specificity: it hydrolyzes peptide bonds at the site of paired basic amino acids and is active in a wide range of pH (5.0–8.0). The main biological function of furin as PC consists in activation of functionally important protein precursors. This is accompanied by initiation of cascades of reactions, which lead to appearance of biologically active molecules involved in realization of specific biological functions both in normal and in some pathological processes. The list of furin substrates includes biologically important proteins such as enzymes, hormones, growth/differentiation, receptors, adhesion proteins, plasma proteins. Furin plays an important role in the development of such processes as proliferation, invasion, cell migration, survival, maintenance of homeostasis, embryogenesis, as well as the development of a number of pathologies, including cardiovascular, cancer, and neurodegenerative diseases. Furin and furin-like proprotein convertases are key factors in the realization of the regulatory functions of proteolytic enzymes; the latter is currently considered as the most important function (compared with well recognized protease function in degradation of proteins).
Keywords: furin; domain structure; specificity; MT1-MMP; growth factors; natriuretic peptides

The role of the signaling pathway FGF/FGFR in pancreatic cancer by D. A. Gnatenko; E. P. Kopantsev; E. D. Sverdlov (101-110).
Fibroblast growth factors (FGFs) are signaling molecules with a wide range of actions that are involved in various processes in the body. Specifically for pancreas, FGFs are important for both organogenesis and carcinogenesis. They belong to the factors involved in the interaction between cancer and stromal cells representing a key component in the development of pancreatic cancer. Pathological changes in the FGF/FGFR signaling pathway is a complex process, which depends on type/isoforms of FGF receptors (FGFR) regulating the remodeling effect and subsequent activation of pancreatic cancer cells by FGF. FGFs and their receptors FGFR are considered as potential specific markers and putative targets for treatment of pancreatic cancer.
Keywords: growth factors; cancer; pancreas; master genes

Dopamine D1–D2 receptor heterodimers: A literature review by N. L. Vekshina; P. K. Anokhin; A. G. Veretinskaya; I. Yu. Shamakina (111-119).
The review summarizes current literature data on the structure of heteromeric complexes of dopamine receptors and their possible role in physiological and pathological processes in the brain. It includes analysis of studies on dopamine D1–D2 receptor complexes, their localization in the brain and the functional role. Functionally, these receptor complexes employ a principally different pathway of signal transduction as compared to the parent homomeric receptors. Investigation of dopamine receptor heteromers extends our understanding of the mechanisms of ligand-receptor interaction and opens new opportunities for the development of pharmacological agents for the treatment of psychiatric disorders associated with impaired dopaminergic neurotransmission, particularly, drug dependence.
Keywords: mesolimbic system; dopamine receptors; heteromeric complexes; drug dependence

The study of the role of mutations M182T and Q39K in the TEM-72 β-lactamase structure by the molecular dynamics method by D. S. Shcherbinin; M. Yu. Rubtsova; V. G. Grigorenko; I. V. Uporov; A. V. Veselovsky; A. M. Egorov (120-127).
Synthesis of β-lactamases is one of the common mechanisms of bacterial resistance to β-lactam antibiotics such as penicillins and cephalosporins. The widespread use of antibiotics resulted in appearance of numerous extended-spectrum β-lactamase variants or inhibitor-resistant β-lactamases. In TEM type β-lactamases mutations of 92 residues have been described. Several mutations are functionally important and they determine the extended substrate specificity. However, roles of the most so-called associated mutations, located far from the active site, remain unknown. We have investigated the role of associated mutations in structure of β-lactamase TEM-72, which contains two key mutations (G238S, E240K) and two associated mutations (Q39K, M182T) by means of molecular dynamics simulation. Appearance of the key mutations (in 238 and 240 positions) caused destabilization of the protein globule, characterized by increased mobility of amino acid residues. Associated mutations (Q39K, M182T) exhibited opposite effect on the protein structure. The mutation M182T stabilized, while the mutation Q39K destabilized the protein. It appears that the latter mutation promoted optimization of the conformational mobility of β-lactamase and may influence the enzyme activity.
Keywords: β-lactamase; TEM-1; TEM-72; mutations; thermoresistance; molecular dynamics

The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of superoxide dismutase activity and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first one is based on registration of adrenochrome (a product of adrenaline autoxidation) at 347 nm; the second approach employs nitro blue tetrazolium (NBT) and registration of diformazan (a product of NBT reduction) at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been given. The main approach consists in the use of a pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some individual features in kinetic behavior of adrenaline autoxidation, they are applicable for the superoxide generating system. Performing measurements at 560 nm, the reaction rate can be regulated by lowering concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this is not applicable. These features of the adrenaline autoxidation reaction may be attributed to the multistage process of adrenaline conversion to adrenochrome and also to coupled electron transfer from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions. This results in formation of corresponding radicals detectable by adding NBT.
Keywords: adrenaline (epinephrine); adrenochrome; superoxide; nitro blue tetrazolium; superoxide dismutase

The peptide-based drug cortexin inhibits brain caspase-8 by A. A. Yakovlev; A. A. Lyzhin; L. G. Khaspekov; A. B. Guekht; N. V. Gulyaeva (134-138).
Although the peptide-based drug cortexin is used in clinical practice for a long time, the mechanisms of its action still remain poorly investigated. This underlies importance of elucidation of the molecular mechanisms responsible for the cortexin impact on brain functioning and identification of primary molecular targets. We hypothesized that the neuroprotective effects of cortexin may be associated with its ability to inhibit brain proteases involved in death of brain cells. Results of this study have shown that cortexin can effectively inhibit brain caspase-8. Other investigated proteases (caspase-1, -3, -9, cathepsin B, and calpain) demonstrated either lower sensitivity or lack of sensitivity to inhibition by cortexin. The original protease inhibitory activity of cortexin was also detected in a (more simple in composition) water-soluble fraction of peptides isolated from cortexin. Both cortexin and the isolated peptide fraction prevented glutamate-induced neuronal death in vitro. Thus the neuroprotective effect of cortexin may be associated with direct inhibition of the brain initiator caspase-8.
Keywords: brain; proteases; caspases; peptides; cortexin

The study of the surface layer of 3D-matrices for tissue engineering by V. S. Chernonosova; R. I. Kvon; E. V. Kiseleva; A. O. Stepanova; P. P. Laktionov (139-145).
Matrices fabricated by electrospinning from polycaprolactone solutions with human albumin or gelatin to 1,1,1,3,3,3-hexafluoroisopropanol have been investigated. Microstructure of matriх surface was analyzed using scanning electron microscopy. Protein distribution in the surface layer was studied by modification with N-(2-hydroxyethyl)phenazine and X-ray photoelectron spectroscopy. The protein concentration in the surface layer of matrices was up to 12 times higher than in the initial solution, and the lower the protein concentration in the solution, the higher the relative protein content is on the surface of matrices. During incubation of matrices in aqueous solutions protein concentration in the surface layer decreased by less than 10% during the first 30–60 min and remained at this level for a long time (seven days). Treatment with proteinase K resulted in about one-third decrease in protein concentration in the surface layer. Thus, both methods used in this study are applicable for analysis of the surface layer of materials fabricated by electrospinning from mixtures of synthetic and natural polymers; however, X-ray photoelectron spectroscopy appears to be a more informative and convenient method.
Keywords: 3D-matrices; synthetic and natural polymers; electrospinning; X-ray photoelectron spectroscopy; chemical modification

Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. II. The putative role of G-quadruplex structures in genomic rearrangements by A. M. Varizhuk; A. V. Sekridova; M. V. Tankevich; V. S. Podgorsky; I. P. Smirnov; G. E. Pozmogova (146-153).
Three evolutionary conserved (G-rich) sites of Alu repeats (PQS2, PQS3, and PQS4) could form in vitro stable inter- and intramolecular G-quadruplexes (GQs). Structures and topologies of these GQs were elucidated using spectral methods. The study of self-association of G-rich Alu fragments performed using a FRET-based method revealed dimeric GQ formation from two distally located sites (PQS2)2, (PQS3)2 or PQS2−PQS3 within one extended single stranded DNA. Using DOSY NMR, AFM microscopy and differential CD spectroscopy it has been demonstrated that oligomer PQS4 (folded into a parallel intramolecular GQ) forms stacks of quadruplexes stabilized by stacking interactions of external G-tetrads. Comparative analysis of the properties of various GQs suggests involvement of two universal general mechanisms of GQ-dependent genomic rearrangements: (i) formation of dimeric GQs from fragments of different molecules; (ii) formation of GQ-GQ-stacks from pre-folded intramolecular parallel GQs from different strands. Thus, association of G-rich Alu motifs with sensitivity to double-strand breaks and rearrangements may be attributed not to structural features of G-rich Alu fragments, but also to their high abundance.
Keywords: G-quadruplex DNA; Alu-repeats; dynamics of DNA secondary structures

Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT by D. D. Zhdanov; D. A. Vasina; E. V. Orlova; V. S. Orlova; M. V. Pokrovskaya; S. S. Aleksandrova; N. N. Sokolov (154-165).
Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of β-deletion splice variant was determined during EndoG overexpression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei as well as after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.
Keywords: EndoG; telomerase; hTERT; alternative splicing; CaCo-2

A universal model of inhibition of neuraminidases from various influenza virus strains by a particular inhibitor has been developed. It is based on known 3D structures for neuraminidases from three influenza virus strains (A/Tokyo/3/67, A/tern/Australia/G70C/75, B/Lee/40) and modeling of the 3D structure of neuraminidases from other strains (A/PR/8/34 and A/Aichi/2/68). Using docking and molecular dynamics, we have modeled 235 enzyme-ligand complexes for 185 compounds with known IC50 values. Selection of final variants among three intermediate results obtained for each enzyme-ligand pair and calculation of independent variables for generation of linear regression equations were performed using MM-PBSA/MM-GBSA. This resulted in the set of equations for individual strains and the equations pooling all the data. Thus using this approach it is possible to predict inhibition for neuraminidase from each the considered strains by a particular inhibitor and to predict the range of its action on neuraminidases from various influenza virus strains.
Keywords: influenza virus neuraminidase; inhibitors; computational methods; QSAR

Mechanisms of perioperative corneal abrasions: Alterations in the tear film proteome by E. Yu. Zernii; O. S. Gancharova; I. E. Ishutina; V. E. Baksheeva; M. O. Golovastova; E. I. Kabanova; M. S. Savchenko; M. V. Serebryakova; L. F. Sotnikova; A. A. Zamyatnin Jr.; P. P. Philippov; I. I. Senin (186-193).
Perioperative corneal abrasion is a common ophthalmic complication detectable in patients undergoing general anesthesia. In this study, using experimental perioperative corneal abrasion in animals (rabbits) correlations have been found between development of corneal abrasion and proteomic changes in the tear film. The process of accumulation of pathological changes in the cornea begins after 1 h of general anesthesia while after 3–6 h of general anesthesia clinically manifested abrasions have been recognized. The development of corneal abrasions was associated with different changes in the content of the major proteins of the tear film. Analysis of the tear proteome points to suppressed lachrymal gland functioning, and suggests that serotransferrin, serum albumin and annexin A1 may be applicable as potential tear markers of the ophthalmic complication. The biochemical changes in the tear film included the rapid decrease in total antioxidant activity and activity of superoxide dismutase, as well as the decrease in interleukin-4 and the increase in interleukin-6 content thus indicating development of oxidative and pro-inflammatory responses. These findings suggest that antioxidant and anti-inflammatory therapy is a prospective approach for prevention/treatment of perioperative corneal abrasions. The observed anesthesia-induced effects should be taken into consideration in any study of ocular surface diseases employing anesthetized animals.
Keywords: general anesthesia; corneal abrasion; dry eye; tear proteome; antioxidant activity; pro-inflammatory response

The methylation status of GSTP1, APC, and RASSF1 genes in human prostate cancer samples: Comparative analysis of diagnostic informativeness of MS-HRM and hybridization on the Illumina Infinium HumanMethylation450 BeadChip by L. O. Skorodumova; K. A. Babalyan; R. Sultanov; A. O. Vasiliev; A. V. Govorov; D. Y. Pushkar; E. A. Prilepskaya; S. A. Danilenko; E. V. Generozov; A. K. Larin; E. S. Kostryukova; E. I. Sharova (194-201).
There is a clear need in molecular markers of prostate cancer (PC) that would specify stratification of patients into risk categories and serve as additional parameters to clinical prognostic factors. Altered DNA methylation status is one of molecular processes that occur during PC development. The gene methylation status may be evaluated in laboratory diagnostics by a PCR approach known as means of methylation-sensitive high resolution melting (MS-HRM) curve analysis. This method is particularly promising due to minimal amounts of DNA required for analysis (10 ng). To date, numerous data based on the use of Infinium HumanMethylation450 BeadChip (HM450) technology have been accumulated on DNA methylation in PC samples. However, it remains unclear whether the MS-HRM results are consistent with chip hybridization data. The aim of this study was to perform the comparative analysis of the diagnostic informativeness of MS-HRM and HM450 chip hybridization methods in determination of the methylation status of three genes GSTP1, APC, and RASSF1 in samples obtained from PC patients. The study has shown that using the MS-HRM method it is possible to discriminate PC tumor samples from samples of histologically unaltered prostate tissue by analyzing the methylation status of each gene. Analysis of chip hybridization data confirmed the results obtained by MS-HRM. Differences between tumor tissue and histologically intact tissue obtained from each PC patient in the methylation levels of GSTP1, APC, and RASSF1, determined by MS-HRM or chip hybridization were consistent with each other. Thus, the assessment of the methylation level of gene GSTP1, APC, and RASSF1 using MS-HRM can become the basis for the development of a laboratory test, the specifying diagnosis of prostate cancer.
Keywords: prostate cancer; DNA methylation; HRM; GSTP1; APC; RASSF1