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LABORATORY OF BIOCHEMISTRY OF PROTEOLYSIS



Head of Laboratory:

    Tatyana A. Valueva, Dr. Sci., Laureate of Russian Federation State premium in 2002 year for the study Chemistry of macromonomers and polymeric hydrogels contained natural physiological active substances.
    tel.:  +7(495)-952-1384
    e-mail: valueva AT inbi.ras.ru

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Staff of Laboratory:

    Gerasimova N.G., PhD.
    Gvozgeva E.L., PhD.
    Ievleva E.V., PhD.
    Kladnitskaya G.V., PhD.
    Kudryavtseva N.N., PhD.
    Revina T.A., PhD.

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Main scientific interests:

    The study of proteolytic enzymes from phytopathogenic microorganisms and their natural plant inhibitors, mechanisms of regulation of plant proteolysis processes and physiological functions of proteinases and their inhibitors during pathogenesis.
    We worked also at the creation of some preparations for biotechnology and medicine on the base of proteinases and their natural inhibitors.

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Main achievements:

         Inhibitors of serine proteinases of the new variety of potato tubers (Solanum tuberosum L. cv. Jubilee Zhukov) table appointments, now adapted and grown in the Moscow region have been studied during 2010-2012. This variety of potato is characterized by low starch content (14%). In addition, potato plants of this variety have increased resistance to viral diseases and infection by the phytopathogenic microorganisms. In this regard, of particular interest is the study of protein inhibitors that are present in tubers of this potato variety and their role in enhancing its nutritional value, and in the processes of the plant defense. A protein designated as PKCI (potato Kunitz-type chymotrypsin inhibitor) have been isolated and purified to homogeneity from potato tubers. Protein purification was performed using the method of gel chromatography on Sephadex G-75 and ion-exchange chromatography on CM-Sepharose CL-6B. The PKCI protein with the same degree of efficiency inhibits the activity of chymotrypsin and trypsin, forming equimolar complexes with the enzymes. It had acted much weaker on subtilisin Carlsberg. It is shown that the protein contains two independent reactive centers of binding for trypsin and chymotrypsin. The N-terminal 20-amino acid sequence of the protein PKCI, which allowed taking it to the family PKPI group B (potato Kunitz-type proteinase inhibitors). The PKCI protein inhibited the growth and development of pathogenic microorganisms such as Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary, causing the potato blight and Fusarium wilt, respectively. It inhibited not only the germination of mycelium, but also accelerated the destruction zoospores and macroconidia of these pathogens. It is highly likely may testify in favor of the fact that the protein plays a role in the defense system of potatoes.
         An mRNA was isolated from the two-week-old seedlings of potato. A cDNA was obtained using this mRNA as a template, using the reversible transcription. Gene-specific primers were designed based on the sequences of genes encoding proteins of the family PKPI Group B in the six varieties of potatoes, which are represented in gene bank NBCI Blast. The specific forward and reverse primers were synthesized. Gene fragments were obtained by PCR with these specific primers. Clones carrying inserts were isolated and sequenced. Two clones with different sequences of full-length cDNA were selected. One of them consisted of 579 bp and encodes a protein containing 193 amino acid residues, and the other - 555 bp and encodes a protein containing only 185 residues. In this case, the second amino acid sequence was reduced in the C-terminal part of the molecule. In this regard, the first cDNA was chosen for further analysis and heterologous expression. Analysis of the 3 'region of genes encoding proteins of the three groups of structural proteins PKPI, suggested that a dedicated cDNA, designated PKPIJ-B, a protein belonging to the group PKPI-B. Analysis of this sequence is allowed to suggest that it encodes a protein PKCI, is present in the tubers. Evaluation of sequence similarity PKPIJ-B cDNA and genes encoding proteins PKPI-B in seven varieties of potato showed that they contain from 89 to 99% identical bp. It turned out that the most variable parts of sequences located at the 5 'end of the molecule. Analyzing the situation nucleotide substitutions in these sequences, we can conclude that they are located in the same sites. It is very likely that recombination occurred in these sites.
         A heterologous expression of cDNA PKPIJ-B was performed in Escherichia coli cells. Plasmid providing heterologous expression of the protein encoded by this cDNA, was created on the basis of the vector pET23a. Expected expression product, which is a recombinant protein PKPIJ-B, was found mainly in inclusion bodies. A method of purification of this protein was developed, including FPLC-chromatography on Mono Q, and subsequent refolding of the recombinant protein. Disk-electrophoresis results showed that the recombinant protein was homogeneous. The PKPIJ-B protein efficiently inhibited the activity of trypsin and chymotrypsin, but had no effect on subtilisin Carlsberg and plant cysteine proteinase (papain). It also contains two independent reactive binding sites of trypsin and chymotrypsin. Thus, there is every reason to believe that the isolated protein was identical to the protein PKCI, isolated from potato tubers. A complete amino acid sequence of the protein PKCI was reconstructed. Analysis of this sequence allowed us to establish the structure and location of the reactive centers in the inhibitor molecule.
         It is shown that changes in available food sources in growth medium phytopathogenic fungus Rhizoctonia solani Kuhn. leads to change not only the quantity produced proteinases, but and their nature and specificity of action. Mineral nitrogen source suppressed the secretion of proteinases by fungus cultured in medium containing heat-stable potato proteins but organic source of nitrogen, along with the acceleration of the growth of mycelium increased their secretion. Based on analysis of the substrate specificity of the extracellular proteinases of the fungus we found that the presence in the culture medium heat-stable potato proteins induced secretion mainly trypsin-like proteinases, and adding to them the proteins of yeast extract subtilisin-like proteinases, while suppressing the production of trypsin-like enzymes. Apparently, in an environment rich in organic nitrogen source, filamentous fungi R. solani loses pathogenicity and becomes saprophyte.
         The hemosorbent "Ovosorb" used to remove proteinases from the blood have been created and put into practice together with Institute of A.V.Topchiev Institute of Petrochemical Synthesis RAS. Approaches to the creation of insulin for oral administration are developing.

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Collaboration:

         The studies on a collaboration linkage grant of the NATO-Russia Joint Scientific and Technological Cooperation Committee are carried out together with the colleagues of Prof. A. Santinos Lab. in ISPA Institute of Sciences of Food Productions, National Researches Council of Italy (Via Montteroni, 73100 Lecce, Italy).
         The work under the agreement of scientific cooperation RAS and NAS Belarus on the topic Isolation, purification, properties, and possible practical application of protein proteinase inhibitors from most promising plant species Compositae are carried out together with the colleagues in V.F. Kuprevich Institute of Experimental Botany NAS Belarus

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Grants:

    The studies on a collaboration linkage grant of the NATO-Russia Joint Scientific and Technological Cooperation Committee are carried out together with the colleagues of Professor A. Santinos Labjratory in ISPA Institute of Sciences of Food Productions, National Researches Council of Italy (Via Montteroni, 73100 Lecce, Italy): Prof. A. Santino, Ph. D., D. P. Poltronieri, Ph. D., D. Fasano. The works of the synthesis of affinity sorbents are carried out together with the colleagues of Laboratory of the Chemistry of Medico-Biological Polymers in A.V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences: Professor L.I. Valuev, Ph. D., D. Chem. Sci., D. L.V. Vanchugova, Ph. D., D. G.A. Sytov, Ph. D.
    The grant of the Russian Foundation for Basic Researches (project No 04-04-48644) The potato subtilisin inhibitors: interaction with exoproteinases of phytopathogenic fungi and their function during pathogenesis (2004-2006 years).
    The grants of the Russian Foundation for Basic Researches (project No 98-04-48191) The study of the structure and the induction mechanism of plant protein proteinase inhibitor (1998-1999 .), (project No 01-04-48072) The study potato tuber Kunitz-type serine proteinase inhibitors: molecular cloning of genes and the induction mechanism during pathogenesis (2001-2002 .).The grant of the NATO-Russia Joint Scientific and Technological Cooperation (project No JSTC.RCLJ.98102) Establishing an universal system for detection of Kunitz-type proteinase inhibitor genes in potato (2004-2005 years)
    The grant of the Russian Foundation for Basic Researches (project No 07-04-00254) The potato Kunitz-type trypsin inhibitors and their functions during an infection by phytopathogenic microorganisms (2007-2009 years)

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Principal Publications (for last 5 years):

    1. Revina T.A., Gerasimova N.G., Kladnitskaya G.V., Chalenko G.I., Valueva T.A. Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms.Applied Biochemistry and Microbiology. 2008. 44, 1, 89-92.
    2. Mosolov V.V., Valueva T.A. Proteinase inhibitors in plant biotechnology: a review. Applied Biochemistry and Microbiology. 2008. 44, 3, 233-240.
    3. Valueva T.A., Speranskaya A.S., Revina T.A., Shevelev A.B. Molecular cloning and expression of genes of Kunitz-type C protease inhibitors from potato. Russian Journal Bioorganic Chemistry. 2008. 34, 3, 319-317.
    4. Valuev L.I., Sytov G.A., Starosel'tseva L.K., Valuev I.L., Vanchugova L.V., Valueva T.A., Ul'yanova M.V., Plate N.A. Oral insulin preparation for regulation of the blood glucose level. Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry. 2010. 4, 1, 113-116.
    5. Revina T. A., Kladnitskaya G. V., Gerasimova N. G., Gvozdeva E. L., Valueva T. A. Protein trypsin inhibitor from potato tubers. Biochemistry (Moscow). 2010. 75, 1, 36-40.
    6. Valueva T. A., Valuev I. L., Obydennova I. V., Valuev L. I. Chemical modification of proteins by "smart" polymers. Russian Journal of Bioorganic Chemistry. 2010, 36, 6, 704707.
    7. Kudravtseva N. N., Gvozdeva E. L., Sof'in A. V., Valueva T. A. The influence of cultural medium composition on the proteolytic enzyme secretion of fungus Rhizoctonia solani. Applied Biochemistry and Microbiology. 2010, 46, 3, 324330.
    8. Revina T. A., Parfenov I. A., Gvozdeva E. L., Gerasimova N. G., Valueva T. A. Chymotrypsin and trypsin inhibitor isolated from potato tubers. Applied Biochemistry and Microbiology. 2011, 47, 3, 239244.
    9. Parfenov I. A., Revina T. A., Pashkovsky P. P., Radyukina N. L., Valueva T. A. Fragment of the gene encoding chymotrypsin and trypsin inhibitor protein of potato tubers. Applied Biochemistry and Microbiology. 2011, 47, 4, 361365.
    10. Mosolov V. V., Valueva T. A. Inhibitors of proteolytic enzymes under abiotic stresses in plants. Applied Biochemistry and Microbiology. 2011, 47, 5, 453459.
    11. Valueva T.A., Kudryavtseva N.N., Revina T.A., Sof'in A.V., Gvozdeva E.L., Ievleva E.V. Comparative analyses of exoproteinases produced by three phytopathogenic microorganisms. J. Pathogens. 2011. 2011. doi:10.4061/2011/947218.
    12. Valueva T.A., Parfenov I.A., Pevina T.A., Morozkina E.V., Benevolensky S.V. Structure and properties of the potato chymotrypsin inhibitor. Plant Physiol. Biochem. 2012, 52, 1, 83-90.

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Last review: 12, December, 2012
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