Department of functioning of living systems

All publications (show selected)

Olga Doncova

H3K36 Methylation and the Chromodomain Protein Eaf3 Are Required for Proper Cotranscriptional Spliceosome Assembly

Laboratory of membrane bioenergetics

Spliceosome assembly takes place in the context of the chromatin environment, suggesting that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin affects splicing, however, are still unclear. Here, we show a role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, the effect of H3K36 methylation on pre-mRNA splicing is mediated through the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the precatalytic B complex around the time of splicing activation, thus revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an “adapter protein” to support efficient, constitutive splicing. Leung et al. demonstrate that H3K36 trimethylation facilitates efficient pre-mRNA splicing through the association of chromodomain protein Eaf3. Eaf3 binds to methylated H3K36 at intron-containing genes to stabilize association of the splicing factor Prp45 and regulate proper cotranscriptional spliceosome assembly.

Publications

  1. Leung CS, Douglass SM, Morselli M, Obusan MB, Pavlyukov MS, Pellegrini M, Johnson TL (2019). H3K36 Methylation and the Chromodomain Protein Eaf3 Are Required for Proper Cotranscriptional Spliceosome Assembly. Cell Rep 27 (13), 3760–3769.e4

Knockdown of the α5 chain of laminins is associated with partial differentiation of tumor cells

Laboratory of microfluidic technologies for biomedicine

The interaction of tumor cells with the components of the extracellular matrix can effect on the rate of disease progression and metastasis. The most important components of this matrix are laminins, heterotrimeric glycoproteins consisting of one α-, one β- and one γ-chain (αβγ). Laminins are involved in the regulation of all the most important processes of living cell by interacting with receptors on the cell surface. In our work, we studied the effect of endogenous expression of the α5 laminin chain on colorectal cancer cells. Knockdown of the α5 chain has been shown to be associated with partial dedifferentiation of tumor cells, apparently by changing the activity of the Wnt signaling pathway and the mTORC1 complex. In addition, ER stress signaling pathways were activated in the cells, which contributed to an increase in the sensitivity of cells to 5-fluorouracil.

Figure: Scheme of the changes that occurred in colon cancer cells in response of the endogenous expression of the α5 laminin chain. Proteins for which there is a decrease or increase in the expression of their genes are highlighted in yellow and green, respectively.

Molecular tools for stabilizing non-canonical nucleic acids secondary structures

The group of molecular tools for living system studies

Ligands and nucleic base modifications for stabilizing non-canonical nucleic acids secondary structures have been developed.

Publications

  1. Schönrath I, Tsvetkov VB, Zatsepin TS, Aralov AV, Müller J (2019). Silver(I)-mediated base pairing in parallel-stranded DNA involving the luminescent cytosine analog 1,3-diaza-2-oxophenoxazine. J Biol Inorg Chem 24 (5), 693–702
  2. (book) Zatsepin TS, Varizhuk AM, Dedkov VG, Shipulin GA, Aralov AV (2019). Oligonucleotide Primers with G8AE-Clamp Modifications for RT-qPCR Detection of the Low-Copy dsRNA. Methods Mol Biol 1973, 281–297
  3. Tsvetkov VB, Zatsepin TS, Turaev AV, Farzan VM, Pozmogova GE, Aralov AV, Varizhuk AM (2019). DNA i-Motifs With Guanidino-i-Clamp Residues: The Counterplay Between Kinetics and Thermodynamics and Implications for the Design of pH Sensors. Comput Struct Biotechnol J 17, 527–536

Benzothiazole-based cyanines as fluorescent “light-up” probes for duplex and quadruplex DNA

The group of molecular tools for living system studies

Analogs of benzothiazole orange (BO) with one, two or three methylbenzothiazolylmethylidene substituents in the 1-methylpyridinium ring were obtained from the respective picolinium, lutidinium or collidinium salts. Fluorescence parameters of the known and new dyes in complexes with various DNA structures, including G-quadruplexes (G4s) and i-motifs (IMs), were analyzed. All dyes efficiently distinguished G4s and ss-DNA. The bi- and tri-substituted derivatives had basically similar distributions of relative fluorescence intensities. The mono-substituted derivatives exhibited enhanced sensitivity to parallel G4s. All dyes were particularly sensitive to a G4 structure with an additional duplex module (the thrombin-binding aptamer TBA31), presumably due to a distinctive binding mode (interaction with the junction between the two modules). In particular, BO showed a strong (160-fold) enhancement in fluorescence quantumyield in complex with TBA31 compared to the free dye. The fluorescence quantum yields of the 2,4-bisubstituted derivative in complex with well-characterized G4s from oncogene promoters were in the range of 0.04e0.28, i.e. comparable to those of ThT. The mono/bi-substituted derivatives should be considered as possible light-up probes for G4 formation.

Publications

  1. Turaev AV, Tsvetkov VB, Tankevich MV, Smirnov IP, Aralov AV, Pozmogova GE, Varizhuk AM (2019). Benzothiazole-based cyanines as fluorescent “light-up” probes for duplex and quadruplex DNA. Biochimie 162, 216–228

Integrator is a key component of human telomerase RNA biogenesis

Laboratory of molecular oncology

Telomeres are special DNA-protein structures that are located at the ends of linear eukaryotic chromosomes. The telomere length determines the proliferation potential of cells. Telomerase is a key component of the telomere length maintenance system. While telomerase is inactive in the majority of somatic cells, its activity determines the clonogenic potential of stem cells as a resource for tissue and organism regeneration. Reactivation of telomerase occurs during the process of immortalization in the majority of cancer cells. Telomerase is a ribonucleoprotein that contains telomerase reverse transcriptase and telomerase RNA components. The RNA processing mechanism of telomerase involves exosome trimming or degradation of the primary precursor. Recent data provide evidence that the competition between the processing and decay of telomerase RNA may regulate the amount of RNA at the physiological level. We show that termination of human telomerase RNA transcription is dependent on its promoter, which engages with the multisubunit complex Integrator to interact with RNA polymerase II and terminate transcription of the human telomerase RNA gene followed by further processing.

Publications

  1. Rubtsova MP, Vasilkova DP, Moshareva MA, Malyavko AN, Meerson MB, Zatsepin TS, Naraykina YV, Beletsky AV, Ravin NV, Dontsova OA (2019). Integrator is a key component of human telomerase RNA biogenesis. Sci Rep 9 (1), 1701
  2. Konopacki C, Pritykin Y, Rubtsov Y, Leslie CS, Rudensky AY (2019). Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function. Nat Immunol 20 (2), 232–242

Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation

Laboratory of membrane bioenergetics

Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133+ PN signature, transition to a CD109+ MES subtype in a C/EBP-β-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133−/CD109+ cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM.

Publications

  1. Minata M, Audia A, Shi J, Lu S, Bernstock J, Pavlyukov MS, Das A, Kim SH, Shin YJ, Lee Y, Koo H, Snigdha K, Waghmare I, Guo X, Mohyeldin A, Gallego-Perez D, Wang J, Chen D, Cheng P, Mukheef F, Contreras M, Reyes JF, Vaillant B, Sulman EP, Cheng SY, Markert JM, Tannous BA, Lu X, Kango-Singh M, Lee LJ, Nam DH, Nakano I, Bhat KP (2019). Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation. Cell Rep 26 (7), 1893–1905.e7

Transcription factor Foxp1 plays important role in Treg

Laboratory of molecular oncology

Regulatory T cells (Treg) is subpopulation of T helper lymphocytes which possesses immune suppressive properties. Treg are critical in protection from excessive immune response and autoimmunity. Unique functions of Treg are defined by transcription factor Fop3 that controls Treg-specific expression of genes. Dr. Yury Rubtsov from the Dept. of functioning of living systems in cooperation with colleagues from the Memorial Sloan-Kettering Cancer Center (New York City) studied a role of Foxp3-related protein, Foxp1, specifically in the murine Treg. In the absence of Foxp1, Treg displayed distorted Foxp3 chromatin binding which led to decreased sensitivity of the cells to IL-2 and diminished suppressive capacity. Results of the study are published in Nature Immunology.

Publications

  1. Konopacki C, Pritykin Y, Rubtsov Y, Leslie CS, Rudensky AY (2019). Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function. Nat Immunol 20 (2), 232–242

Antiviral compounds based on 5-(perylen-3-ylethynyl)uracil scaffold

Group of Cross-Linking Enzymes,  Laboratory of molecular design and synthesis

Rigid amphipathic fusion inhibitors (RAFIs) are potent antivirals based on a perylene core linked with a nucleoside moiety. Sugar-free analogues of RAFIs, 5-(perylen-3-ylethynyl)uracil-1-acetic acid and its amides were synthesized using combined protection group strategy. Compounds appeared to have low toxicity on porcine embryo kidney (PEK) or rhabdomiosarcoma (RD) cells together with remarkable activity against enveloped tick-borne encephalitis virus (TBEV): EC50 values vary from 0.077 μM to subnanomolar range. Surprisingly, 3-pivaloyloxymethyl (Pom) protected precursors showed even more pronounced activity. The propargylamide of N3-Pom-protected 5-(perylen-3-ylethynyl)uracil acetic acid, a universal precursor, was used in CuAAC click reaction for the synthesis of several derivatives including three ramified molecules with high activities against tick borne encephalitis virus (TBEV). Pentaerythritol-based polyazides were used for the assembly of molecules containing 2…4 antiviral 5-(perylen-3-ylethynyl)uracil scaffolds, the first examples of polyvalent perylene antivirals. Four prepared compounds, including one ramified cluster, showed remarkable 1…3 nM EC50 values against TBEV in cell culture. The results suggest that the carbohydrate moiety of RAFI nucleosides does not play a crucial role in their antiviral action, and biological activity of the 5-(perylen-3-ylethynyl)uracil scaffold can be effectively modulated by substituents in positions 1 and 3. The high antiviral activity of new compounds, coupled with low toxicity advocate their potential role in antiviral therapy. The antiviral evaluations were performed in Chumakov Institute of Poliomyelitis and Viral Encephalitides.

Fluorescent Yin-Yang probes for RT-qPCR detection of low copy HIV RNA

Laboratory of molecular design and synthesis

Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and molecular diagnostics. We synthesized novel DNA molecular beacons double labeled with the identical dyes (R6G, ROX and Cy5) at 5′- and 3′-end, studied their photophysical properties, and demonstrated that fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity (LLQ: 10 vs 30 copies mL-1) in comparison to commercially available Abbott RealTime HIV-1 kit based on VIC-BHQ dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples). The research was performed in collaboration with ScolTech, Research Institute of Epidemiology, and other institutions.

Publications

  1. (book) Kireev DE, Farzan VM, Shipulin GA, Korshun VA, Zatsepin TS (2020). RT-qPCR Detection of Low-Copy HIV RNA with Yin-Yang Probes. Methods Mol Biol 2063, 27–35
  2. Farzan VM, Kvach MV, Aparin IO, Kireev DE, Prikazchikova TA, Ustinov AV, Shmanai VV, Shipulin GA, Korshun VA, Zatsepin TS (2019). Novel homo Yin-Yang probes improve sensitivity in RT-qPCR detection of low copy HIV RNA. Talanta 194, 226–232

Ultrasharp probes for biomolecules atomic force microscopy

Group of electron microscopy

During the last years electron microscopy group has been working on the efficient use of atomic force microscopy for visualization of biomacromolecules. Previously, we have determined empirically the conditions for obtaining nanostructures on the tips of standard atomic force microscope probes, which improve the resolution of the method by an order of magnitude (from 15-20 nm to ~ 2 nm). In 2018, we completed a study of the physical properties of these structures, explaining the growth mechanisms and efficiency in microscopy. According to the results of work in 2018, an article was published in the journal Ultramicroscopy, included in 1 quartile. In 2017, 2 papers were presented at conferences.

Synthesis takes place using the method of gas-phase chemical deposition, activated by plasma. Statistical analysis of electron microscopy images shows that the growth rate of branched nanostructures is about 100 nm / hour. The tip radius of the obtained structures is 1-2 nm. The results of the study of the probes obtained by the method of Raman scattering show that the branched structures consist of amorphous carbon, apparently, similar to glassy carbon, since they have high mechanical characteristics.

Publications

  1. Obraztsova EA, Basmanov DV, Barinov NA, Klinov DV (2018). Carbon Nanospikes: Synthesis, characterization and application for high resolution AFM. Ultramicroscopy 197, 11–15

New antiviral nucleoside derivatives for inhibiting the reproduction of varicella-zoster virus and tick-borne encephalitis virus

Laboratory of molecular design and synthesis,  The group of molecular tools for living system studies

A series of analogues of potent antiviral perylene nucleoside dUY11 with methylthiomethyl (MTM), azidomethyl (AZM) and HO-C1-4-alkyl-1,2,3-triazol-1,4-diyl groups at 3`-O-position as well as the two products of copper-free alkyne-azide cycloaddition of the AZM derivative were prepared and evaluated against tick-borne encephalitis virus (TBEV). Four compounds showed EC50 ≤10 nM, thus appearing the most potent TBEV inhibitors to date. Moreover, these nucleosides have higher lipophilicity (clogP) and increased solubility in aq. DMSO vs. parent compound dUY11.

Phenoxazine scaffold is widely used to stabilize nucleic acid duplexes, as a part of fluorescent probes for the study of nucleic acid structure, recognition, and metabolism etc. We present the synthesis of phenoxazine-based nucleoside derivatives and their antiviral activity against a panel of structurally diverse viruses: enveloped DNA herpesviruses varicella zoster virus (VZV) and human cytomegalovirus, enveloped RNA tick-borne encephalitis virus (TBEV), and non-enveloped RNA enteroviruses. Studied compounds were effective against DNA and RNA viruses reproduction in cell culture. 3-(2’-Deoxy-β-D-ribofuranosyl)-1,3-diaza-2-oxophenoxazine proved to be a potent inhibitor of VZV replication with superior activity against wild type than thymidine kinase deficient strains (EC50 0.06 and 10 µM, respectively). This compound did not show cytotoxicity on all the studied cell lines. Several compounds showed promising activity against TBEV (EC50 0.35-0.91 µM), but the activity was accompanied with pronounced cytotoxicity. These compounds may be considered as a good starting point for further structure optimization as antiherpesviral or antiflaviviral compounds.

Publications

  1. Proskurin GV, Orlov AA, Brylev VA, Kozlovskaya LI, Chistov AA, Karganova GG, Palyulin VA, Osolodkin DI, Korshun VA, Aralov AV (2018). 3′-O-Substituted 5-(perylen-3-ylethynyl)-2′-deoxyuridines as tick-borne encephalitis virus reproduction inhibitors. Eur J Med Chem 155, 77–83

The structure of the two components of the lipopeptide antibiotic crystallomycin from a sample obtained 60 years ago has been established. The identity of the components of two crystallomycin components to these of aspartocin (the structure of which has been elucidated recently) has been found. The antibiotic exhibits Ca2+ -dependent activity against gram-positive bacteria. The conformations of crystallomycin 2 in solution were investigated using NMR.

The amino acid 4-chloro-L-kinurenin, previously found in natural products only once, was found in the peptide antibiotic INA-5812. We first described the fluorescent properties of 4-chloro-L-kinurenin and its use as an energy donor for the excitation of other fluorophores.

The structure of two new macrolide antibiotics, astolides A and B, has been established using various 2D NMR techniques. Astolide molecules contain simultaneously a membrane-active polyol macrolide and a redox-active naphthoquinone residue as aglycones. The presence of a hydroxyl group at position 18 dramatically changes the spectrum of biological activity in comparison with the known analogues – antifungal activity increases and cytotoxicity reduces.

Publications

  1. Alferova VA, Shuvalov MV, Suchkova TA, Proskurin GV, Aparin IO, Rogozhin EA, Novikov RA, Solyev PN, Chistov AA, Ustinov AV, Tyurin AP, Korshun VA (2018). 4-Chloro-l-kynurenine as fluorescent amino acid in natural peptides. Amino Acids 50 (12), 1697–1705
  2. Alferova VA, Novikov RA, Bychkova OP, Rogozhin EA, Shuvalov MV, Prokhorenko IA, Sadykova VS, Kulko AB, Dezhenkova LG, Stepashkina EA, Efremov MA, Sineva ON, Kudryakova GK, Peregudov AS, Solyev PN, Tkachev YV, Fedorova GB, Terekhova LP, Tyurin AP, Trenin AS, Korshun VA (2018). Astolides A and B, antifungal and cytotoxic naphthoquinone-derived polyol macrolactones from Streptomyces hygroscopicus. Tetrahedron 74 (52), 7442–7449
  3. Jiang ZK, Tuo L, Huang DL, Osterman IA, Tyurin AP, Liu SW, Lukyanov DA, Sergiev PV, Dontsova OA, Korshun VA, Li FN, Sun CH (2018). Diversity, novelty, and antimicrobial activity of endophytic actinobacteria from mangrove plants in Beilun Estuary National Nature Reserve of Guangxi, China. Front Microbiol 9 (MAY), 868
  4. Tyurin AP, Alferova VA, Paramonov AS, Shuvalov MV, Malanicheva IA, Grammatikova NE, Solyev PN, Liu S, Sun C, Prokhorenko IA, Efimenko TA, Terekhova LP, Efremenkova OV, Shenkarev ZO, Korshun VA (2018). Crystallomycin revisited after 60 years: Aspartocins B and C. Medchemcomm 9 (4), 667–675

The role of protein regulators of splicing in the intercellular communication of tumor cells

Group of Cross-Linking Enzymes,  Laboratory of membrane bioenergetics

A novel mechanism of communication between tumor cells mediated by intercellular transport of spliceosomes has been demonstrated. Investigation of clinical samples from patients with ovarian cancer, as well as primary cultures of human glioblastoma cells, allowed us to observe that, upon induction of apoptosis, many tumor cells secrete into the extracellular environment various components of the spliceosome, a ribonucleoprotein complex that splices RNA molecules in eukaryotes. During the early stages of apoptosis the spliceosomes are transported from the nucleus to the cytoplasm, and then are secreted inside membrane vesicles (exosomes). These vesicles can be captured by neighboring tumor cells, and the splicing proteins contained therein affect mRNA splicing in the recipient cells. This leads to the formation of different protein isoforms and the acquisition of a more aggressive and treatment-resistant phenotype of surviving tumor cells. One of the key players of this process was identified as the RBM11 protein, which is readily transferred between glioblastoma cells and regulates alternative splicing of important oncogenes such as MDM4 and CyclinD1.