Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.11, #3)

Analysis of the role of protein phosphorylation in the development of diseases by M. G. Zavialova; V. G. Zgoda; E. N. Nikolaev (203-218).
During recent decades significant progress in studies of the molecular basis of socially significant diseases has been achieved due to introduction of high-throughput methods of genomics and proteomics. Numerous studies, performed within the global program “Human Proteome,” were aimed at identifying all possible proteins in various (including cancer) cell cultures and tissues. One of the aims was to identify socalled biomarkers—the proteins, specific for certain pathologies. However, many studies have shown that the development of the disease is not associated with appearance of new proteins, but it depends on the expression level of certain genes or specific proteoforms representing splice variants, single amino acid polymorphism (SAP) and post-translational modifications (PTM) of proteins. PTMs can play a key role in the development of pathology, because they activate various regulatory or structural proteins in most cellular processes. Among such modifications, phosphorylation appears to be the most significant PTM. This review considers methods of analysis of protein phosphorylation used in studies of the molecular basis of oncological diseases; it contains examples illustrating contribution of modified proteins directly involved in their development as well as examples of screening of such crucial PTMs in diagnostics and selection of methods for treatment.
Keywords: proteomics; cancer; neurodegenerative diseases; post-translational modifications (PTM); phosphorylation

Suppression of telomerase activity in leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase by M. V. Pokrovskaya; D. D. Zhdanov; M. A. Eldarov; S. S. Aleksandrova; A. V. Veselovsky; V. S. Pokrovskiy; D. V. Grishin; Ju. A. Gladilina; N. N. Sokolov (219-233).
Active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA) (RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T) have been obtained by the method of site-directed mutagenesis. The variants RrAE149R, V150P, F151T and RrА+N17, E149R, V150P could decrease reduce expression of the hTERT telomerase subunit and, therefore, activity of telomeres in Jurkat cells, but not in cellular lysates. At the same time, L-asparaginasеs of Escherichia coli, Erwinia carotovora, and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K did not suppress telomerase activity. It is suggested that some regions in the RrA structure (amino acids residues 146–164, 1–17, 60–67) are responsible for suppression of telomerase activity. The results obtained show that antineoplastic activity of some RrA variants is associated both with reduction of concentration of free L-asparagine, and with decreased expression of the hTERT telomerase subunit; this opens new prospects for antineoplastic therapy.
Keywords: L-asparaginase Rhodospirillum rubrum ; antineoplastic effect of L-asparaginase; a site-directed mutagenesis; telomerase; hTERT subunit of telomerase

Hepatic differentiation of adult and fetal liver stromal cells in vitro by I. V. Kholodenko; R. V. Kholodenko; G. V. Manukyan; K. N. Yarygin (234-242).
The liver has a marked capacity for regeneration. In most cases the liver regeneration is determined by hepatocytes. The regenerative capacity of hepatocytes is significantly reduced in acute or chronic damage. For example, in patients with alcoholic cirrhosis repair mechanisms are not activated and only organ transplantation or advanced methods of regenerative medicine can help such patients. Clinical trials including patients with various forms of liver disease have shown promising results of transplantation of autologous bone marrow stem cells. However, improvement of the effectiveness of such treatment requires optimization of sources of progenitor cells. In this study we have isolated stromal cells from the liver biopsies of three patients with alcoholic cirrhosis, performed their morphological and phenotypic analysis, and evaluated the hepatic potential of these cells in vitro. Stromal cells isolated from the fetal liver were used for comparative evaluation. During hepatic differentiation both types of cells expressed hepatic markers and secreted albumin. These results can serve as a basis for the development of a new method for the treatment of end-stage liver disease. The stromal cells isolated from the liver biopsies proliferate for a long time in a culture and this provides opportunity to produce them in large amounts for subsequent differentiation into hepatocyte-like cells and autologous transplantation.
Keywords: adult liver stromal cells; fetal liver stromal cells; hepatic differentiation; alcoholic cirrhosis; hepatocyte- like cells

Morphofunctional changes of dendritic cells induced by sulfated polysaccharides of brown algae by I. D. Makarenkova; N. K. Akhmatova; S. P. Ermakova; N. N. Besednova (243-250).
The effect of structurally different sulfated polysaccharides from brown algae Fucus evanescens, Saccharina cichorioides, and Saccharina japonica on the morphofunctional changes of dendritic cells has been studied using flow cytometry and phase-contrast microscopy. It has been found that the dendritic cells are characterized by larger sizes, vacuolated cytoplasm, eccentrically located nucleus, the presence of numerous cytoplasmic pseudopodia of various forms; they do express surface markers, thus providing evidence for their maturation (CD83, CD11c, HLA-DR, CD86). Increased production of immunoregulatory (IL-12) and proinflammatory cytokines (TNF-α, IL-6) by dendritic cells polarizes the development of the Th-1 type immune response.
Keywords: dendritic cells; sulfated polysaccharides; morphology; fucoidans; cytokines

Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes by D. D. Zhdanov; D. A. Vasina; E. V. Orlova; V. S. Orlova; V. S. Pokrovsky; M. V. Pokrovskaya; S. S. Aleksandrova; N. N. Sokolov (251-264).
Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated expression of the active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.
Keywords: cisplatin; EndoG; telomerase; hTERT; alternative splicing; malignant transformation

Contamination of exosome preparations, isolated from biological fluids by A. E. Grigor’eva; N. S. Dyrkheeva; O. E. Bryzgunova; S. N. Tamkovich; B. P. Chelobanov; E. I. Ryabchikova (265-271).
The aim of this study was to attract attention of researchers to the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 (JEOL, Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: blood plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and conditioned cell culture medium (MDCK, MDA-MB, and MCF-7 cells). All examined preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations were characterized by the presence of contaminating structures: low electron density particles without limiting membrane and therefore could not be attributed to exosomes (“non-vesicles”). Two main types of the “non-vesicles” were found in the exosome preparations: particles of 20–40 nm in size, representing 10–40% of all structures in the exosome preparations; and particles of 40–100 nm in size (identical to exosomes by size). Morphology of the “non-vesicles” corresponded to that of intermediate and low density lipoproteins (20–40 nm), and very low density lipoproteins (40–100 nm), which were identical to exosomes in their size. The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparations by electron microscopy and to take into consideration the presence of contaminating structures in the analysis of experimental data.
Keywords: exosomes; electron microscopy; lipoproteins

Novel agonists of melatonin receptors as promising hypotensive and neuroprotective agents for therapy of glaucoma by N. B. Chesnokova; O. V. Beznos; N. A. Lozinskaya; M. S. Volkova; E. V. Zaryanova; N. S. Zefirov; A. V. Grigoryev (272-278).
Melatonin is a pineal hormone that has a capacity to lower intraocular pressure; it exhibits neuroprotective and antioxidant properties that make it possible to use melatonin in the therapy of glaucoma. Melatonin analogs demonstrating affinity to melatonin receptors are promising candidates for application as antiglaucomatous agents. Chemical modification of the melatonin structure can increase efficiency, bioavailability, and selectivity of melatonin analogs. We have designed and synthesized a number of new 2-oxindole derivatives, the ligands of melatonin MT3 receptors; these analogs are characterized by the ability to lower intraocular pressure in normotensive rabbits and high antioxidant activity against hydroxyl radical and superoxide anion-radical. New ligands significantly exceeding melatonin in antioxidant activity can be also applicable for the development of therapeutic agents for treatment of oxidative stress. The maximal hypotensive effect of the analogs was comparable to and lasted longer than that of melatonin. Combination of these properties suggests potential used of the analyzed melatonin analogs in complex therapy of glaucoma.
Keywords: melatonin; melatonin analogs; melatonin receptors; intraocular pressure; antioxidant activity; glaucoma

Determination of EGFR gene somatic mutations in tissues and plasma of patients with non-small cell lung cancer by O. I. Brovkina; M. G. Gordiev; A. N. Toropovskiy; D. S. Khodyrev; R. F. Enikeev; O. A. Gusev; L. H. Shigapova; A. G. Nikitin (279-285).
Activating mutations in the EGFR gene influence cell proliferation, angiogenesis, and increases metastatic ability of non-small cell lung cancer (NSCLC) cells; they have a significant impact on the choice of medical therapy of NSCLC. The use of targeted therapy with tyrosine kinase inhibitors requires performance of appropriate genetic tests in NSCLC patients. The aim of this study was to develop a real-time PCRbased diagnostic test-system for rapid and cost-effective analysis of EGFR mutations in paraffin blocks and plasma and to perform comparative estimation of diagnostic characteristics features of real-time wild type blocking PCR and digital PCR. The study included 156 patients with different degrees of lung adenocarcinoma differentiation. A simple and efficient real-time PCR-based method for detection of L858R activating mutation and del19 deletion in the EGFR gene in DNA isolated from paraffin blocks or blood has been developed. The test system for EGFR mutations has been validated using 411 samples of paraffin blocks. The proposed system demonstrated high efficiency for DNA testing from paraffin blocks: a concordance with results of testing by means a Therascreen® EGFR RGQ PCR Kit (Qiagen, Germany) was 100%. Applicability of this test system has been also demonstrated for detection of mutations in plasma.
Keywords: EGFR gene; activating mutations; real-time PCR; non-small cell lung cancer; plasma

Nitrobenzofuroxane derivatives as dual action HIV-1 inhibitors by S. P. Korolev; M. A. Pustovarova; A. M. Starosotnikov; M. A. Bastrakov; Yu. Yu. Agapkina; S. A. Shevelev; M. B. Gottikh (286-290).
Human immunodeficiency virus type 1 (HIV-1) causes one of the most dangerous diseases, HIV infection and AIDS. The search for new inhibitors of the virus still remains an urgent task. One of approaches to suppress the HIV infection is the use of dual action HIV-1 inhibitors, i.e. inhibitors targeting two stages of the viral life cycle. The catalytic domain of HIV-1 integrase shares similar structural organization with the ribonuclease (RNase H) domain of HIV-1 reverse transcriptase, and therefore an approach aimed at creation of dual action inhibitors which would simultaneously inhibit HIV-1 integrase and RNase H seems to be very promising. In this work we have synthesized a series of 6-nitrobenzofuroxane derivatives and studied their inhibitory activity towards two HIV-1 enzymes, integrase and RNase H.
Keywords: HIV-1; inhibitor; integrase; reverse transcriptase; RNase H

Isolation and characterization of exosomes from blood plasma of breast cancer and colorectal cancer patients by S. N. Tamkovich; N. V. Yunusova; M. N. Stakheeva; A. K. Somov; A. E. Frolova; N. A. Kiryushina; S. G. Afanasyev; A. E. Grigor’eva; P. P. Laktionov; I. V. Kondakova (291-295).
A simple approach for isolation of exosomes from blood plasma samples has been proposed. Using this approach it is possible to obtain highly purified preparations of microvesicles no larger than 100 nm. The presence of different subpopulations of exosomes isolated by this method has been recognized in the blood plasma of healthy donors and cancer patients. Universal markers CD9, CD24, and CD81 are applicable for routine typing of exosomes isolated from blood plasma samples.
Keywords: exosomes; transmission electron microscopy; flow cytometry; colorectal cancer; breast cancer

Redox-dependent mechanisms of regulation of breast epithelial cell proliferation by E. A. Stepovaya; E. V. Shakhristova; O. L. Nosareva; E. V. Rudikov; M. Yu. Egorova; D. Yu. Egorova; V. V. Novitsky (296-300).
Activation of free radical oxidation in various types of cells, including breast epithelial cells, can lead to damage to macromolecules, particularly proteins involved in regulation of proliferation and programmed cell death. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. In this regard, modulation of the redox status of cells by means of a blocker and a protector of SH-groups of proteins can be used as a model for studying the role of redox proteins and glutathione in regulation of cell proliferation during the development of various pathological processes. In this study the state of thioredoxin, glutaredoxin, glutathione systems and their role in the regulation of HBL-100 breast epithelial cell proliferation during modulation of the redox status by using N-ethylmaleimide (NEM) and 1,4-dithioerythritol (DTE) have been investigated. The modulation of the redox status of the breast epithelial cells by the blocker (NEM) and the protector (DTE) of thiol groups of proteins and peptides influenced the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase by changing concentrations of GSH and GSSG. Modulation of the redox status of HBL-100 cells was accompanied by an increase in the number of cells in the S phase of the cell cycle and a decrease of cells in G0/G1 and G2/M phases as compared with the intact cell culture. The proposed method for evaluating the proliferative activity of cells during modulation of their redox state can be used in the development of new therapeutic approaches for treatment of diseases accompanied by the development of oxidative stress.
Keywords: thioredoxin; glutaredoxin; glutathione; oxidative stress; proliferation; breast epithelial cells

Molecular mechanisms of antitumor activity of the polymeric form of niclosamide with respect to human colorectal cancer cells by A. S. Zhirnik; Yu. P. Semochkina; E. Yu. Moskaleva; N. I. Krylov; I. A. Tubasheva; S. L. Kuznetsov; E. A. Vorontsov (301-307).
Using poly(lactic-co-glycolic) acid a polymeric form of niclosamide (PFN) has been developed and its antitumor activity against human colorectal cancer cell lines SW837, Caco-2, COLO 320 HSR has been investigated in comparison with free niclosamide. PFN was shown to be more cytotoxic against cancer cells and less cytotoxic against normal cells (human embryonic lung fibroblasts) as compared to niclosamide. Free niclosamide and PFN share a common mechanism of the cytotoxic action on tumor cells, which is associated with mitochondrial damage (evaluated as a decrease in rhodamine 123 accumulation), and increased levels of reactive oxygen species, particularly mitochondrial superoxide anion, causing oxidative damage of intracellular targets. The action of niclosamide and PFN was accompanied by G0/G1 cell cycle arrest.
Keywords: niclosamide; polymeric form of niclosamide; poly(lactic-co-glycolic) acid; reactive oxygen species; cell cycle; colorectal cancer