Laboratory of Reparation Enzymes

Georgy Nevinsky,
prof., D.Sc
The Russia State Prize in Science and Technique (1999)
Phone: +7(383)363-51-26 Email

Molecular mechanisms of protective and repairing systems in prokaryotes and humans:

• I. Studies of molecular mechanisms of viral, pro- and eukaryotic enzymes involved in DNA replication, repair, and dynamics (integration etc.).
• II. Studies of molecular mechanisms and biological roles of human and animal immunoglobulins with enzyme-like properties.

Main scientific results Direction I:

• With help of method of stepwise increase of the ligand complexity (SILC)developed in our laboratory, steady-state and fast kinetics, chemical and affine modifications as well as other physicochemical methods general regularities of DNA specific and nonspecific recognition by topoisomerase, HIV integrase, depurine-, depyrimidine endonuclease, uracyl-DNA-glycosylase and human 8-oxoguanin-DNA-glycosylase as well as E. coli 8-oxoguanin-DNA-glycosylase and lactofferin were determined on quantitative level. The relative contribution of weak additive nonspecific interactions, specific contacts, the stage of DNA and enzyme mutual adaptation as well as the catalytic stage in supplying enzymes with high affinity to DNA and high specificity its action was identified. All enzymes were described with complete thermodynamic and kinetic models. [Biochemistry. 2003. 42. 9235; Nucleic Acids Res. 2004. 32. 5134; Eur. Biophys. J. 2005. 34, 541; FEBS J. 2005. 272. 2734; FEBS Lett. 2006. 580, 4916; J. Biol. Chem. 2007. 282, 1029; Biochem. Biophys. Res. Commun. 2008. 368, 175; FEBS J. 2008. 275, 3747; FEBS Lett. 2008. 582, 410; J. Mol. Recognit. 2011. 24, 656; Nucleic Acids Res. 2011. 39, 4836; J. Mol. Recognit. 2013. 26, 136].
• Principles of accumulation of oxidative DNA damage in cells of liver, lungs and different parts of brain as well as dynamics of changes of repair enzymes and enzymes with antioxidant properties in organs of rapidly senescent rats were ascertained. New oxidizers effectively protected cells of different organs including brain from oxidative stress conducting to development of oncological and different pathologies of middle age were found. Series of new polyfluorinated derivatives of 1,4-naphthoquinone with a low toxicity to ordinary cells but a high activity of growth inhibition of human cancer cells were revealed. [Kemeleva et al., Mutat. Res. 2006. 599, 88; Biokhimiya (Moscow). 2006. 71, 760; Bioorgan. Khimiya (Moscow), 2008. 34, 558; Biochim. Biophys. Acta. 2013. Feb 9; Zakharova O.A. et al., Eur. J. Med. Chem. 2010. 45, 270; Eur. J. Med. Chem. 2010. 45, 2321; Bioorg. Med. Chem. 2011. 19, 256; Patent RF N.2387635, 2009; Patent RF N.2443678, 2010].
• Lactoferrin (LF), the protein of acute phase and nonspecific protection from harmful factors of environment, was shown for the first time to be the enzyme possessing five different enzymatic activities: DNase, RNase, ATPase, phosphatase and hydrolyzing of polysaccharide. Сonsistent patterns of recognition of DNA by LF were determined. [Kanyshkova et al., FEBS Lett. 1999. 451, 235; Kanyshkova et al., Eur. J. Biochem. 2003. 270, 3353 ; Nevinskii A.G., J. Mol. Recogn. 2009. 22, 330; Soboleva S.E. et al., Mol. Biol. (Moscow). 2009. 43, 157; Guschina et al., J. Mol. Recognit. 2013. 26(3), 136].

Direction II:

• We have discovered that blood from patients with some autoimmune pathologies (multiple sclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis, polyarthritis, hepatitis, AIDS, tick-borne encephalitis) contains IgG with RNase and DNase activity. The titer of antibodies and relative level of specific activity of IgGs in DNA hydrolysis may be used as a proxy to follow the disease progress and the efficiency of different treatment regimes. [Nevinsky G.A. et al., In book: “Protein-protein interactions. A molecular cloning manual”. NY, 2002, 523; Nevinsky G.A., Buneva V.N. In book: “Catalytic antibodies”. 2005, 505; Nevinsky G.A. In: Autoimmune Diseases: Symptoms, Diagnosis and Treatment. 2010, 1; Nevinsky G.A., Buneva V.N., ScientificWorldJournal. 2010. 10, 1203; Nevinsky G.A. In book: Understanding HIV/AIDS Management and Care - Pandemic Approaches in the 21st Century. InTech. 2011, 151; Buneva V.N. et al., Biokhimiya (Moscow). 2003. 68, 1088].
• It was shown that the development of autoimmune diseases (AID) associated with production of abzymes with different activities was connected with change of level of proliferation and a differentiation of cells of marrow of mice, and also level of proliferation of lymphocytes in various bodies, depending on a stage of AID, pregnancy and lactation. [Andryushkova A.A. et al., FEBS Lett. 2006; J. Cell. Mol. Med. 2007. 11, 531; Int. Immunol. 2009. 21, 935; Nevinsky G.A., Buneva V.N., Biokhimiya (Moscow). 2009. 74, 1165].
• The post-transmitting modification of IgG and sIgA in vitro from human milk connected with half-molecule exchange between milk Ig of various subclasses (but not free light and heavy chains) was shown for the first time provided affinity polyspecificity and catalytic polyreactivity of natural immunoglobulins. [Nevinsky G.A. et al., PLoS One. 2012. 7, e42942; Sedykh S.E. et al., PLoS ONE. 2012. 7, e48756].
• Abzymes specifically hydrolyzing the (MBP) which was the main component of a myelin – a protein and lipid cover of axons that leads to violation of carrying out nervous impulses were found for the first time in blood of patients with multiple sclerosis (MS) and systemic lupus erythematosus (SLE). The main properties and regularities of functioning of these abzymes were ascertained. All sites of proteolysis of MBP molecule by abzymes were determined. [Polosukhina et al., J. Cell Mol. Med. 2004. 8, 359; Med. Sci. Monit., 2005. 1, BR266; Immunol. Lett., 2006. 103, 75; Legostaeva et al., J. Cell Mol. Med., 2010. 14, 699; Bezuglova et al., J. Mol. Recognit. 2011. 24, 960; Int. Immunol. 2012. 24, 759; Peptides. 2012. 37, 69].
• It was shown that preparations of polyclonal IgG and IgM from HIV-infected blood of patients contained fractions of antibodies which, unlike classical proteases, with high efficiency hydrolyze only HIV integrase, but not other proteins. It was ascertained that small abzyme fractions at HIV infection function on various mechanisms: tiol-, acid-, serine-like, metal-dependent proteases which hydrolyze this virus protein on about forty sites that leads to suppression of a catalysis integrase reactions 3’-processing and integration. [Baranova et al., Biochimie. 2009. 91, 1081; Int. Immunol. 2010. 22, 671; Odintsova et al., J. Mol. Recognit. 2011. 24, 1067; Baranova S.V. et al., Biokhimiya (Moscow). 2011. 76, 1300; Odintsova et al., J. Mol. Recognit. 2012 25, 193; Int. Immunol. 2011. 23, 601; J. Mol. Recognit. 2013. 26, 121; Nevinsky G.A. et al., Patent RF N.2396278, 2010].

1. Parkhomenko TA, Doronin VB, Castellazzi M, Padroni M, Pastore M, Buneva VN, Granieri E, Nevinsky GA. Comparison of DNA-Hydrolyzing Antibodies from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis. A, Krasitskaya VV, Fokina AA, Timoshenko VV, Nevinsky GA, Venyaminova AG, Frank LA. RNA Aptamer against autoantibodies associated with multiple sclerosis and bioluminescent detection probe on its basis. Anal Chem. 2014. 86(5):2590-4.
2. Kostrikina I.A., Buneva V.N., Nevinsky G.A. Systemic lupus erythematosus: Molecular cloning of fourt1. Parkhomenko TA, Doronin VB, Castellazzi M, Padroni M, Pastore M, Buneva VN, Granieri E, Nevinsky GA. Comparison of DNA-Hydrolyzing Antibodies from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis. PLoS One. 2014. 9(4):e93001.
3. Vorobjeva M.A., Krasitskaya V.V., Fokina A.A., Timoshenko V.V., Nevinsky G.A., Venyaminova A.G., Frank L.A. RNA Aptamer against autoantibodies associated with multiple sclerosis and bioluminescent detection probe on its basis. Anal Chem. 2014. 86(5):2590-4.
4. Kostrikina I.A., Buneva V.N., Nevinsky G.A. Systemic lupus erythematosus: Molecular cloning of fourteen recombinant DNase monoclonal kappa light chains with different catalytic properties. Biochim. Biophys. Acta - General Subjects. 2014. 1840(6), 1725-1737.
5. Botvinovskaya A.V., Kostrikina I.A., Buneva V.N., Nevinsky G.A. Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties. J. Mol. Recognit. 2013. 26(10), 450-460.
6. Sattarova E. A., Sinitsyna O.I., Vasyunina E.A., Duzhak T.G., Kolosova N.G., Zharkov D.O., Nevinsky G.A. Age-dependent guanine oxidation in DNA of different brain regions of Wistar rats and prematurely aging OXYS rats. Biochim. Biophys. Acta, General Subjects. 2013. 1830(6), 3542-3552.
7. Bezuglova A.M., Dmitrenok P.S., Konenkova L.P., Buneva V.N., Nevinsky G.A. Multiple Sites of the Cleavage of 21- and 25-Mer Encephalytogenic Oligopeptides Corresponding to Human Myelin Basic Protein (MBP) by Specific Anti-MBP Antibodies from Patients with Systemic Lupus Erythematosus. PloS ONE. 2013. 8(3), e51600.
8. Odintsova E.S., Dmitrenok P.S., Buneva V.N., Nevinsky G.A.Specific anti-integrase abzymes from HIV-infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20-mer oligopeptides corresponding to its antigenic determinants. J. Mol. Recognit. 2013. 26(3), 121-135.
9. Gushina T.A., Soboleva S.E., Nevinsky G.A. Recognition of specific and nonspecific DNA by human lactoferrin. J. Mol. Recognit. 2013. 26(3), 136-148.
10. Kundzer A.V., Volkova M.V., Bogdanos D.P., Rödiger S., Schierack P., Generalov I., Nevinsky G.A., Roggenbuck D. Deoxyribonuclease activity of polyclonal IgGs: a putative serological marker in patients with spondyloarthritides. Immunologic Research. 2013. 56(2-3), 457-464.
11. Legostaeva G.A., Zaksas N., Gluhcheva Y., Sedykh S.E., Madzharova M., Atanassova N., Buneva V.N., Nevinsky G.A. Effect of CoCl2 on the content of different metals and a relative activity of DNA-hydrolyzing abzymes in the blood plasma of mice. J. Mol. Recognit. 2013. 26(1), 10-22.
12. Nevinsky G.A., Buneva V.N., Sedykh S.E. Human Milk IgGs Contain Various Combinations of Different Antigen-Binding Sites Resulting in Multiple Variants of their Bispecificity. PloS ONE. 2012. 7(8), e42942.
13. Sedykh S.E., Buneva V.N., Nevinsky G.A. Human Milk sIgA Molecules Contain Various Combinations of Different Antigen-Binding Sites Resulting in a Multiple Binding Specificity of Antibodies and Enzymatic Activities of Abzymes. PloS ONE. 2012. 7(11), e48756.
14. Bezuglova A.M., Dmitrenok P.S., Konenkova L.P., Buneva V.N., Nevinsky G.A. Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus. Peptides. 2012. 37(1), 69-78.
15. Bezuglova A.M., Konenkova L.P., Buneva V.N., Nevinsky G.A. IgGs Containing Light Chains of the lambda- and kappa- type and of all Subclasses (IgG1-IgG4) from the Sera of Patients with Systemic Lupus Erythematosus Hydrolyze Myelin Basic Protein. Int. Immunol. 2012. 24(12), 759-770.
16. Odintsova E.S., Baranova S.V., Dmitrenok PS, Calmels C, Parissi V, Andreola ML, Buneva V.N., Nevinsky G.A. Anti-integrase abzymes from the sera of HIV-infected patients specifically hydrolyze integrase but nonspecifically cleave short oligopeptides. J. Mol. Recognit. 2012. 25(4), 193-207.
17. Parkhomenko T.A., Buneva V.N., Doronin B.M., Volkova M.V., Senkovich S.A., Generalov I.I., Nevinsky G.A. IgGs containing λ- and κ-type light chains and of all subclasses (IgG1-IgG4) from the sera of patients with autoimmune diseases and viral and bacterial infections hydrolyze DNA. J. Mol. Recognit. 2012. 25(7), 383-392.
18. Nevinsky G.A., Buneva V.N. Autoantibodies and Natural Catalytic Antibodies in Health, Multiple Sclerosis, and Some Other Diseases. Advances in Neuroimmune Biology. 2012. 3(2), 157-182.
19. Labetskii V.S., Dmitrenok P.S., Buneva V.N., Nevinsky G.A. Minor oligosaccharides strongly bound with abzymes of human milk. Rus. J. Immunol. (Moscow). 2012. 6(1), 22-34.
20. Kirpota O.O., Endutkin A.V., Ponomarenko M.P., Ponomarenko P.M., Zharkov D.O., Nevinsky G.A. Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase. Nucleic Acids Res. 2011. 39, 4836-4850.
21. Odintsova E.S., Baranova S.V., Dmitrenok P.S., Rasskazov V.A., Calmels C., Parissi V., Andreola M.L., Buneva V.N., Zakharova O.D., Nevinsky G.A. Antibodies to HIV integrase catalyze site-specific degradation of their antigen. Int. Immunol. 2011. 23, 601-612.
22. Kostrikina I.A., Kolesova M.E., Orlovskaya I.A., Buneva V.N., Nevinsky G.A. Diversity of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL/MPJ-lpr mice. J. Mol. Recognit. 2011. 24, 557-569.
23. Nevinsky G.A. Structural, thermodynamic, and kinetic basis for the activities of some nucleic acid repair enzymes. J. Mol. Recognit. 2011. 24, 656-677.
24. Zakharova O.D., Ovchinnikova L.P., Goryunov L.I., Troshkova N.M., Shteingarts V.D., Nevinsky G.A. Cytotoxicity of new polyfluorinated 1,4-naphtoquinones with diverse substituents in the quinone moiety. Bioorg. Med. Chem. 2011. 19, 256-260.

Research is supported by Grants of the Russian Foundation for Basic Research (RFBR), Siberian Branch of Russian Academy of Sciences and Program of RAS Presidium «Molecular and Cell Biology».

Electrophoresis and blotting systems (separation of proteins and nucleic acids); incubators and readers for ELISA (Bio-Rad); cell culture equipment (laminar-flow cabinet, СО2-incubator, microscope); analytical FPLC and HPLC (PerSeptive Biosystems BioCad, Waters Breeze 2).