V S Prasolov

Институт молекулярной биологии им. В.А. Энгельгардта Российской академии наук, Moskva, Moscow, Russia

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Publications (63)21.84 Total impact

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    ABSTRACT: Recently we reported benzohydroxamic acids (BHAs) as potent and selective inhibitors of hepatitis C virus (HCV) replicon propagation. In this work 12 pyridine hydroxamic acids (PHAs) were synthesized and tested in full-genome replicon assay. It was found that PHAs possessed very similar anti-HCV properties compared to BHAs. Both classes of hydroxamic acids caused hyperacetylation of α-tubulin pointing to inhibition of histone deacetylase 6 (HDAC6) as part of their antiviral activity. The tested compounds did not inhibit the growth of poliovirus, displaying high selectivity against HCV. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Bioorganic & medicinal chemistry letters 04/2015; 25(11). DOI:10.1016/j.bmcl.2015.04.016 · 2.33 Impact Factor
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    ABSTRACT: The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.
    01/2015; 7(1):87-97.
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    ABSTRACT: Two novel mutations in the glucokinase gene (GCK) have been identified in patients with maturity-onset diabetes of the young type-2 (MODY2), i.e., a C-for-G substitution at position −1 of the acceptor splice site of intron 7 (c. 864-1G>C) and a synonymous c.666C>G substitution (GTC>GTG, p.V222V) at exon 6. An analysis of the splicing products obtained upon the transfection of human embryonic HEK293 cells with GCK minigene constructs carrying these mutations showed that both substitutions impaired normal splicing. As a result of c.864-1G>C, the usage of the normal acceptor site was blocked, which activated cryptic acceptor splice sites within intron 7 and generated several aberrant RNAs containing fragments of intron 7. The synonymous substitution c.666C>G created a novel donor splice site in exon 6, which results in the formation of an abnormal GCK mRNA with a 16-nucleotide deletion in exon 6. In vitro experiments on minigene splicing confirmed the inactivating effect of these mutations on glucokinase gene expression.
    Molecular Biology 03/2014; 48(2):248-253. DOI:10.1134/S0026893314020071 · 0.74 Impact Factor
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    ABSTRACT: Currently, neutron capture therapy is a promising cancer treatment. This method is based on the reaction of thermal neutron capture by some nonradioactive elements (e.g., Gd157), which results in the sub-sequent emission of electrons and gamma rays. An effective instrument for delivering gadolinium into tumor tissue are “rigid” nanostructures (NSs) based on double-stranded DNA complexes with gadolinium (NS-Gd). The local concentration of Gd in these nanostructures may reach 40%. To optimize the process of neutron capture therapy, it is very important to investigate possible mechanisms of the penetration of NS-Gd particles into tumor cells. In this work, the dynamics of interaction between NS-Gd and cultivated Chinese hamster ovary cells (CHO) was studied by confocal and electron microscopy. NS-Gd were shown to be able to enter CHO cells. This process started after about 1 h of incubation. After 6 h, NS-Gd particles were detected in almost all cells. A further increase in the incubation time did not lead to significant changes in cell morphology, although the amount of NS-Gd inside cells continued to increase. The plasma membranes of the cells remained intact. Once entering the cells, NS-Gd particles remained there for a long time. The data show that NS-Gd has relatively low toxicity and suggest that the presence of NS-Gd in tumor cells does not prevent their division. The data are important for improving the efficiency of the method of neutron-capture therapy.
    Molecular Biology 09/2013; 47(5):743-750. DOI:10.1134/S0026893313050178 · 0.74 Impact Factor
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    ABSTRACT: A diverse collection of 40 derivatives of benzohydroxamic acid (BHAs) of various structural groups were synthesized and tested against hepatitis C virus (HCV) in full-genome replicon assay. Some of these compounds demonstrated an exceptional activity, suppressing viral replication at sub-micromolar concentrations. The compounds were inactive against key viral enzymes NS3, and NS5B in vitro assays, suggesting host cell inhibition target(s). The testing results were consistent with metal coordination by the BHAs hydroxamic group in complex with a target(s). Remarkably, this class of compounds did not suppress poliomyelitis virus (PV) propagation in RD cells indicating a specific antiviral activity of BHAs against HCV.
    Bioorganic & medicinal chemistry letters 08/2013; 23(21). DOI:10.1016/j.bmcl.2013.08.081 · 2.33 Impact Factor
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    ABSTRACT: Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.
    Molecular Biology 01/2013; 47(1). DOI:10.1134/S0026893313010123 · 0.74 Impact Factor
  • Doklady Biochemistry and Biophysics 01/2013; 448(1):49-51. DOI:10.1134/S1607672913010134 · 0.37 Impact Factor
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    ABSTRACT: The effect of sulfated polysaccharides on the efficiency of infection of mouse embryonic fibroblast cell lines SC-1 and NIH-3T3 by replication-competent recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the eGFP gene was investigated. It was shown that used polysaccharides have no cytostatic and cytotoxic effects on SC-1 and NIH 3T3 cells inthe concentrations from 0.01 to 100 μg/ml and have virucidal activity against Mo-MuLV. Polysaccharides in the indicated concentrations inhibit cell infection by Mo-MuLV, that prevents further expansion of viral infection. It was detected that sulfated polysaccharides are effective inhibitors of other retroviruses, including lentiviruses, that use heparan sulfate as cell receptors for non-specific binding.
    Molekuliarnaia biologiia 05/2012; 46(3):508-18. DOI:10.1134/S0026893312030119
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    ABSTRACT: Hyperexpression of oncogene c-kit is found in 80% patients with acute myeloid leukemia (AML). The transgenic model cell line expressing the oncogene c-kit was obtained by transduction with recombinant retrovirus. We have designed small interfering RNAs (siRNA) efficiently suppressing the expression of activated oncogene c-kit. Further small hairpin RNAs (shRNA) targeting c-kit mRNA were designed and expressed in lentiviral vectors. We report a stable reduction in c-kit expression following the introduction of shRNAs into model cells as well as Kasumi-1 cells from the patient with AML.
    Molekuliarnaia biologiia 12/2011; 45(6):1036-45. DOI:10.1134/S0026893311060136
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    ABSTRACT: Novel mutation in CYP21A2 gene causing the steroid 21-hydroxylase deficiency - C to G substitution in 7-position ofintron 2 acceptor splice site (c.290-7C>G) was identified. The effect of the mutation on splicing was checked in the system of CYP21A minigene expression in the cultured mammalian cells. The mutation impairs the usage of intron 2 acceptor splice site resulting in intron retention.
    Bioorganicheskaia khimiia 11/2011; 37(6):815-20. DOI:10.1134/S1068162011060124
  • A E Vilgelm, A I Zaika, V S Prasolov
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    ABSTRACT: First time p53 was found in the complex with viral large T-antigene in the cells transformed by small DNA virus SV40. The cloning of p53 cDNA was done in the beginning of eighties and soon after that the whole p53 gene was cloned. The p53 family is comprised of three genes: TP53,TP63 and TP73, each of which is expressed as a set of structurally and functionally different isoforms. All of them intensively interact with each other forming a united functional network of proteins. In this review we discuss evolution of the p53 family and significance of all its members in embryonic development, reproduction, regeneration, regulation of aging and life span, as well as in the body's defense against cancer. With special attention we review the role of less studied members of the p53 family: p63 and p73, in oncogenesis and tumor progression and show that different isoforms of these proteins might exert a contrary effect on these processes.
    Molekuliarnaia biologiia 02/2011; 45(1):180-97. DOI:10.1134/S002689331101016X
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    ABSTRACT: In the present study we have applied the siRNA approach for substantial reduction of AML1-ETO and RUNX1 (K83N) expression, which are frequently found in the leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5'-untranslated region of mRNA for the mutant RUNX1 (K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of the oncogenes following the introduction of shRNAs into cells.
    Molekuliarnaia biologiia 10/2010; 44(5):876-88. DOI:10.1134/S0026893310050146
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    ABSTRACT: Baculovirus expression vectors are extensively used for the delivery of foreign genes and expression of recombinant proteins in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient transient and stable transduction of diverse mammalian cells ensure a high level of heterologous protein expression both in vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter, green or red fluorescent protein gene, SV40pA polyadenylation signal, and polylinker MCS were constructed for the delivery of genes encoding hepatitis C virus structural proteins into mammalian cells. In HEK293T and Huh7 cells, formation of glycoprotein complexes and HCV4ike particles was observed. A high efficiency of the baculovirus-medi-ated gene transfer and expression of the virus envelope proteins in mammalian cells was demonstrated using fluorescence, flow cytometry, and immunoblot techniques. Key wordsbaculovirus AcMNPV-BacMam virus-hepatitis C virus (HCV)-HCV structural proteins-HCV-like particles-mammalian cells HEK293T-COS-7 and Huh7-Sf9 insect cells
    Molecular Biology 06/2010; 44(3):479-487. DOI:10.1134/S0026893310030180 · 0.74 Impact Factor
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    ABSTRACT: Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.
    Molecular Biology 06/2009; 43(3):403-409. DOI:10.1134/S002689330903008X · 0.74 Impact Factor
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    ABSTRACT: Our aim was to investigate how replication protein A (RPA) in a wide range of concentration can regulate the activity of human telomerase. We used an in vitro system based on human cell extracts with or without RPA. It has been shown that removal of RPA leads to loss of telomerase activity and addition of RPA restores telomerase activity and at the same time promotes telomerase processivity. However, high excess of RPA inhibited telomerase processivity and caused the synthesis of relatively short DNA fragments (about 50-100 nucleotides). We assume that, together with other telomere-binding proteins, RPA may take part in activation of telomere overhang elongation by telomerase at a certain stage of a cell cycle as well as in regulation of telomere length.
    Biochemistry (Moscow) 02/2009; 74(1):92-6. DOI:10.1134/S0006297909010143 · 1.35 Impact Factor
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    ABSTRACT: An enzymatic assay system was developed to quantify the distribution of recombinant proteins over various cell structures. The system takes advantage of α-complementation of β-galactosidase. The large ω fragment of β-galactosidase is expressed in predefined cell structures with the aid of attached protein localization signals. The resulting reporter cell lines are infected with a second construct expressing a target protein fused with the shorter α fragment of β-galactosidase. The physical proximity of the two recombinant proteins carrying the β-galactosidase fragments results in the reconstitution of an active enzyme, and its activity is measured with a plate reader. The recombinant constructs are based on lentiviral vectors and can be rapidly and efficiently introduced into cells by infection with stocks of lentivirus particles. The efficiency of the system was demonstrated with the FOXO3A transcription factor, which shuttles between the cytoplasm and nucleus in the model colon carcinoma cell line RKO.
    Molecular Biology 12/2008; 42(6):894-900. DOI:10.1134/S0026893308060095 · 0.74 Impact Factor
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    ABSTRACT: Bicyclic furano[2,3-d]pyrimidine ribonucleosides were synthesized by Pd(0)- and CuI-catalyzed coupling of 5-iodouridine with terminal alkynes. The treatment of the resulting nucleosides with ammonia or methylamine solution in aqueous alcohol resulted in pyrrolo- and N(7)-methylpyrrolo[2,3-d]pyrimidine nucleosides. 5'-O-Triphosphates of bicyclic nucleosides were obtained by the treatment of the nucleosides with POCl3 in the presence of a "proton sponge." The 5'-O-triphosphates are not substrates for HCV RNA-dependent RNA polymerase, but are effective substrates for HCV RNA helicase/NTPase and did not inhibit ATP hydrolysis. Only 3-(beta-D-ribofuranosyl)-6-decyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one showed a moderate anti-HCV activity in the HCV replicon system and efficiently inhibited replication of bovine viral diarrhea virus (BVDV) in KCT-cells, other compounds being inactive. None of the compounds were cytotoxic within the tested range of concentrations.
    Bioorganicheskaia khimiia 09/2008; 34(5):661-70. DOI:10.1134/S1068162008050099
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    ABSTRACT: The long 5-untranslated region (5'-UTR) of the human retrotransposon L1 harbors a unique internal promoter which ensures new copies of this mobile element to be much less dependent on an integration site at the level of transcription. The mechanism of this promoter's action still remains unclear, but due to some early studies the opinion has been -formed that the most important part for its function ("minimal promoter") is the first 100-150 nts of the 5'-UTR. In this paper we show that activity of the "minimal promoter" is rather poor in comparison with the entire 5'-UTR. The absolutely crucial part which is indispensable for the effective transcription is the internal region of the 5'-UTR (+390...+662) containing multiple binding sites for various transcription factors. This region may be considered as a transcriptional enhancer. Deletion of this segment leads to a dramatic lost of transcription level irrespectively of cell type, while deletion of the first 100 nt decreases the transcription efficiency no more than 1.5 to 2-fold. Thus, the organization of the L1 regulatory region may be much more similar to that of well-studied invertebrate LINE elements than it was thought before. Also we suggest a possible existence of an alternative sense promoter within the internal part of the L1 5'-UTR driving the synthesis of a 5'-truncated mRNA of the retrotransposon.
    Molekuliarnaia biologiia 01/2007; 41(3):508-14.
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    ABSTRACT: Human gene RFP2 is a candidate tumor suppressor located at 13q14.3 and deleted in multiple tumor types. To explore regulation of RFP2, we determined structure of the 5'-untranslated region of RFP2 gene and its promoter. RFP2 promoter area is TATA-less, highly enriched in G and C nucleotides, and contains multiple quadruplex forming GGGGA-repeats. Deletion analysis of 5'-flanking sequences demonstrated that repeat containing fragment possesses activity seven times exceeding that of the combined SV40 promoter/enhancer. Other unusual features of the RFP2 promoter include anomalously high electrostatic fields induced by sequence-dependent dipoles and very low nucleosome forming potential. A "minimized" version of the RFP2 promoter could be used for overexpression of the various transgenes in the mammalian cells.
    Biochemical and Biophysical Research Communications 05/2006; 342(3):859-66. DOI:10.1016/j.bbrc.2006.01.187 · 2.28 Impact Factor

Publication Stats

108 Citations
21.84 Total Impact Points

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Institutions

  • 1975–2015
    • Институт молекулярной биологии им. В.А. Энгельгардта Российской академии наук
      Moskva, Moscow, Russia
  • 2014
    • Moscow Institute of Physics and Technology
      Moskva, Moscow, Russia
  • 2004–2013
    • Russian Academy of Sciences
      • Institute of Molecular Biology
      Moskva, Moscow, Russia
  • 2003
    • Pacific Institute of Bioorganic Chemistry
      Wladiwostok, Primorskiy, Russia