Говорун Вадим Маркович

Избранные публикации

  1. Babenko V.V., Mikov A.N., Manuvera V.A., Anikanov N.A., Kovalchuk S.I., Andreev Y.A., Logashina Y.A., Kornilov D.A., Manolov A.I., Sanamyan N.P., Sanamyan K.E., Kostryukova E.S., Kozlov S.A., Grishin E.V., Govorun V.M., Lazarev V.N. (2017). Identification of unusual peptides with new Cys frameworks in the venom of the cold-water sea anemone Cnidopus japonicus. Sci Rep 7 (1), 14534 [+]

    Sea anemones (Actiniaria) are intensely popular objects of study in venomics. Order Actiniaria includes more than 1,000 species, thus presenting almost unlimited opportunities for the discovery of novel biologically active molecules. The venoms of cold-water sea anemones are studied far less than the venoms of tropical sea anemones. In this work, we analysed the molecular venom composition of the cold-water sea anemone Cnidopus japonicus. Two sets of NGS data from two species revealed molecules belonging to a variety of structural classes, including neurotoxins, toxin-like molecules, linear polypeptides (Cys-free), enzymes, and cytolytics. High-throughput proteomic analyses identified 27 compounds that were present in the venoms. Some of the toxin-like polypeptides exhibited novel Cys frameworks. To characterise their function in the venom, we heterologously expressed 3 polypeptides with unusual Cys frameworks (designated CjTL7, CjTL8, and AnmTx Cj 1c-1) in E. coli. Toxicity tests revealed that the CjTL8 polypeptide displays strong crustacean-specific toxicity, while AnmTx Cj 1c-1 is toxic to both crustaceans and insects. Thus, an improved NGS data analysis algorithm assisted in the identification of toxins with unusual Cys frameworks showing no homology according to BLAST. Our study shows the advantage of combining omics analysis with functional tests for active polypeptide discovery.

  2. Fesenko I., Khazigaleeva R., Kirov I., Kniazev A., Glushenko O., Babalyan K., Arapidi G., Shashkova T., Butenko I., Zgoda V., Anufrieva K., Seredina A., Filippova A., Govorun V. (2017). Alternative splicing shapes transcriptome but not proteome diversity in Physcomitrella patens. Sci Rep 7 (1), 2698 [+]

    Alternative splicing (AS) can significantly impact the transcriptome and proteome of a eukaryotic cell. Here, using transcriptome and proteome profiling data, we analyzed AS in two life forms of the model moss Physcomitrella patens, namely protonemata and gametophores, as well as in protoplasts. We identified 12 043 genes subject to alternative splicing and analyzed the extent to which AS contributes to proteome diversity. We could distinguish a few examples that unambiguously indicated the presence of two or more splice isoforms from the same locus at the proteomic level. Our results indicate that alternative isoforms have a small effect on proteome diversity. We also revealed that mRNAs and pre-mRNAs have thousands of complementary binding sites for long non-coding RNAs (lncRNAs) that may lead to potential interactions in transcriptome. This finding points to an additional level of gene expression and AS regulation by non-coding transcripts in Physcomitrella patens. Among the differentially expressed and spliced genes we found serine/arginine-rich (SR) genes, which are known to regulate AS in cells. We found that treatment with abscisic (ABA) and methyl jasmonic acids (MeJA) led to an isoform-specific response and suggested that ABA in gametophores and MeJA in protoplasts regulate AS and the transcription of SR genes.

  3. Lomakin Y., Arapidi G.P., Chernov A., Ziganshin R., Tcyganov E., Lyadova I., Butenko I.O., Osetrova M., Ponomarenko N., Telegin G., Govorun V.M., Gabibov A., Belogurov A. (2017). Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo. Front Immunol 8, 777 [+]

    Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

  4. Ziganshin R.H., Ivanova O.M., Lomakin Y.A., Belogurov A.A. Jr, Kovalchuk S.I., Azarkin I.V., Arapidi G.P., Anikanov N.A., Shender V.O., Piradov M.A., Suponeva N.A., Vorobyeva A.A., Gabibov A.G., Ivanov V.T., Govorun V.M. (2016). The pathogenesis of demyelinating form of Guillain-Barre syndrome: proteo-peptidomic and immunological profiling of physiological fluids. Mol. Cell Proteomics , [+]

    Acute inflammatory demyelinating polyneuropathy (AIDP) - the main form of Guillain-Barre syndrome (GBS) - is a rare and severe disorder of the peripheral nervous system (PNS) with an unknown etiology. One of the hallmarks of the AIDP pathogenesis is a significantly elevated cerebrospinal fluid (CSF) protein level. In this paper CSF peptidome and proteome in AIDP were analyzed and compared with multiple sclerosis (MS) and control patients. A total protein concentration increase was shown to be due to even changes in all proteins rather than some specific response, supporting the hypothesis of protein leakage from blood through the blood-nerve barrier. The elevated CSF protein level in AIDP was complemented by activization of protein degradation and much higher peptidome diversity. Due to the studies of the acute motor axonal form, GBS as a whole is thought to be associated with autoimmune response against neurospecific molecules. Thus, in AIDP, autoantibodies against cell adhesion (CAM) proteins localized at Ranvier's nodes were suggested as possible targets in AIDP. Indeed, AIDP CSF peptidome analysis revealed CAM proteins degradation, however no reliable dependence on the corresponding autoantibodies levels was found. Proteome analysis revealed overrepresentation of Gene Ontology groups related to responses to bacteria and virus infections, which were earlier suggested as possible AIDP triggers. Immunoglobulin blood serum analysis against most common neuronal viruses did not reveal any specific pathogen; however AIDP patients were more immunopositive in average and often had polyinfections. Cytokine analysis of both AIDP CSF and blood did not show a systemic adaptive immune response or general inflammation, while innate immunity cytokines were upregulated. To supplement the widely-accepted though still unproven autoimmunity-based AIDP mechanism we propose a hypothesis of the primary PNS damaging initiated as an innate immunity-associated local inflammation following neurotropic viruses egress, while the autoantibody production might be an optional complementary secondary process.

  5. Fesenko I., Seredina A., Arapidi G., Ptushenko V., Urban A., Butenko I., Kovalchuk S., Babalyan K., Knyazev A., Khazigaleeva R., Pushkova E., Anikanov N., Ivanov V., Govorun V.M. (2016). The Physcomitrella patens Chloroplast Proteome Changes in Response to Protoplastation. Front Plant Sci 7, 1661 [+]

    Plant protoplasts are widely used for genetic manipulation and functional studies in transient expression systems. However, little is known about the molecular pathways involved in a cell response to the combined stress factors resulted from protoplast generation. Plants often face more than one type of stress at a time, and how plants respond to combined stress factors is therefore of great interest. Here, we used protoplasts of the moss Physcomitrella patens as a model to study the effects of short-term stress on the chloroplast proteome. Using label-free comparative quantitative proteomic analysis (SWATH-MS), we quantified 479 chloroplast proteins, 219 of which showed a more than 1.4-fold change in abundance in protoplasts. We additionally quantified 1451 chloroplast proteins using emPAI. We observed degradation of a significant portion of the chloroplast proteome following the first hour of stress imposed by the protoplast isolation process. Electron-transport chain (ETC) components underwent the heaviest degradation, resulting in the decline of photosynthetic activity. We also compared the proteome changes to those in the transcriptional level of nuclear-encoded chloroplast genes. Globally, the levels of the quantified proteins and their corresponding mRNAs showed limited correlation. Genes involved in the biosynthesis of chlorophyll and components of the outer chloroplast membrane showed decreases in both transcript and protein abundance. However, proteins like dehydroascorbate reductase 1 and 2-cys peroxiredoxin B responsible for ROS detoxification increased in abundance. Further, genes such as thylakoid ascorbate peroxidase were induced at the transcriptional level but down-regulated at the proteomic level. Together, our results demonstrate that the initial chloroplast reaction to stress is due changes at the proteomic level.

  6. Fesenko I.A., Arapidi G.P., Skripnikov A.Y., Alexeev D.G., Kostryukova E.S., Manolov A.I., Altukhov I.A., Khazigaleeva R.A., Seredina A.V., Kovalchuk S.I., Ziganshin R.H., Zgoda V.G., Novikova S.E., Semashko T.A., Slizhikova D.K., Ptushenko V.V., Gorbachev A.Y., Govorun V.M., Ivanov V.T. (2014). Specific pools of endogenous peptides are present in gametophore, protonema, and protoplast cells of the moss Physcomitrella patens. BMC Plant Biol. 15 (1), 87 [+]

    Protein degradation is a basic cell process that operates in general protein turnover or to produce bioactive peptides. However, very little is known about the qualitative and quantitative composition of a plant cell peptidome, the actual result of this degradation. In this study we comprehensively analyzed a plant cell peptidome and systematically analyzed the peptide generation process.

  7. Shender V.O., Pavlyukov M.S., Ziganshin R.H., Arapidi G.P., Kovalchuk S.I., Anikanov N.A., Altukhov I.A., Alexeev D.G., Butenko I.O., Shavarda A.L., Khomyakova E., Evtushenko E., Ashrafyan L.A., Antonova I.B., Kuznetcov I.N., Gorbachev A.Y., Shakhparonov M.I., Govorun V.M. (2014). Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication. Mol. Cell Proteomics , [+]

    Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in natural environment. This medium is of interest as a promising source of potential biomarkers and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of malignant ascites metabolome and proteome. To omit components that belong to systemic response to the ascites formation we compared malignant ascites with cirrhosis one. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively, 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrated that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest, the finding suggests that they may play a role in the communication between cancer cells. Besides, malignant ascites contains a high number of exosomes that are known to play an important role for signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication.

  8. Osterman I.A., Ustinov A.V., Evdokimov D.V., Korshun V.A., Sergiev P.V., Serebryakova M.V., Demina I.A., Galyamina M.A., Govorun V.M., Dontsova O.A. (2013). A nascent proteome study combining click chemistry with 2DE. Proteomics 13 (1), 17–21 [+]

    To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.

  9. Ziganshin R., Arapidi G., Azarkin I., Zaryadieva E., Alexeev D., Govorun V., Ivanov V. (2011). New method for peptide desorption from abundant blood proteins for plasma/serum peptidome analyses by mass spectrometry. J Proteomics 74 (5), 595–606 [+]

    This report describes a new method for desorption of low-molecular weight (LMW) peptides from abundant blood proteins for use in subsequent mass spectrometry analyses. Heating of diluted blood serum to 98°C for 15min resulted in dissociation of LMW peptides from the most abundant blood proteins. Application of blood plasma/serum fractionation using magnetic beads with a functionalized surface followed by heating of the resultant fractions significantly increases the number of LMW peptides detected by MALDI-TOF MS, enhances the general reproducibility of mass spectrometry profiles and considerably increases the number of identified blood serum peptides by LC-MS/MS using an Agilent 6520 Accurate-Mass Q-TOF.

  10. Ziganshin R.K.h., Arapidi G.P., Azarkin I.V., Balmasova I.P., Timchenko O.L., Fedkina Iu.A., Morozova E.A., Piradov M.A., Suponeva N.A., Iushchuk N.D., Govorun V.M. (2011). [Proteomic technologies for identification of serum potential biomarkers of autoimmune demyelinating polyneuropathies]. Bioorg. Khim. 37 (1), 36–44 [+]

    Time-of-flight MALDI mass spectrometry (MALDI-TOF-MS) profiling of blood serum of patients with Guillain-Barré syndrome (GBS, 36 samples), chronic inflammatory demyelinating polyneuropathy (CIDP, 24 samples) and practically healthy donors (HD) (35 samples) was carried out in order to identify potential biomarkers of autoimmune demyelinating polyneuropathies (ADP). To simplify the peptide-protein mixture of serum prior to MALDI-TOF-MS analysis samples were pre-fractionated on magnetic microparticles with a weak cation-exchange (MB-WCX) surface. Comparative analysis of mass spectrometric data using the classification algorithms (genetic and neural network-controlled) revealed a characteristic set of peaks, agreed change area with a high specificity and sensitivity of the differentiated mass spectrometry profiles of the blood serum of patients with DPNP and healthy donors (for GBS values of these characteristics reached 100 and 100, and for CIDP 94.1 and 100% respectively). Comparative analysis of mass spectrometric profiles of serum samples obtained from patients with GBS and CIDP, allowed to build a classification model to differentiate these diseases from each other, with a specificity of 88.9 and a sensitivity of 80%.

  11. Ziganshin R.K.h., Alekseev D.G., Arapidi G.P., Ivanov V.T., Moshkovskiĭ S.A., Govorun V.M. (2008). [Serum proteome profiling for ovarion cancer diagnosis using ClinProt magnetic bead technique and MALDI-TOF-mass-spectrometry]. Biomed Khim 54 (4), 408–19 [+]

    Using reverse-phase (MB-HIC 8 and HB-HIC 18) weak cation exchange (MB-WCX) and metal affinity ClinProt magnetoc beads peptides and protein factions were obtained from human sera for their profiling by MALDI-TOF mass spectrometry. Proteome profiling of sera from I-IV stage ovarian cancer patients (47 women, average age 51) and from healthy women (47 subjects, average age 49) using MB-WCX beads allowed calculation of the best diagnostic models based on the Genetic Algorithm and Supervised Neural Network classifiers; these model generated 100% sensitivity and specificity when the test set of subjects was analyzed. Introduction of additional sera from patients with colorectal cancer (19) and ulcerous colitis (5) to the statistical model confirmed 100% ovarian cancer recognition. Statistical mass-spectrometry analysis of mass-spectrometry peak areas included to the diagnostic classifiers showed 3 peaks distinctive for ovarian cancer and 4 peaks distinctive for ovarian and colorectal cancer.

  12. Reshetnyak A.V., Armentano M.F., Ponomarenko N.A., Vizzuso D., Durova O.M., Ziganshin R., Serebryakova M., Govorun V., Gololobov G., Morse H.C. 3rd, Friboulet A., Makker S.P., Gabibov A.G., Tramontano A. (2007). Routes to covalent catalysis by reactive selection for nascent protein nucleophiles. J. Am. Chem. Soc. 129 (51), 16175–82 [+]

    Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.

  13. Ponomarenko N.A., Durova O.M., Vorobiev I.I., Belogurov A.A. Jr, Kurkova I.N., Petrenko A.G., Telegin G.B., Suchkov S.V., Kiselev S.L., Lagarkova M.A., Govorun V.M., Serebryakova M.V., Avalle B., Tornatore P., Karavanov A., Morse H.C. 3rd, Thomas D., Friboulet A., Gabibov A.G. (2006). Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen. Proc. Natl. Acad. Sci. U.S.A. 103 (2), 281–6 [+]

    Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.