Institute of Biotechnology

bti8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 260 2103
Fax 260 2116
E-mail:
www.ibt.lt

Director - Prof. Habil. Dr.  Kęstutis Sasnauskas

STAFF

82 research fellows (6 habilitated doctors and 51 doctors), 30 doctoral students.

 

MAIN RESEARCH AREAS OF THE INSTITUTE

Proteins and chemical compounds for biomedicine
Structural biology and bioinformatics
Genomics, biomolecules and biotechnologies: fundamental and applied research

DOCTORAL DISSERTATIONS MAINTAINED IN 2012

Z. Liutkevičiūtė. DNA cytosine methyltransferase-directed reactions involving non-cofactors like compounds.

A. Šilanskas. Restriction endonuclease-triplex forming oligonucleotide conjugates with controllable catalytic activity.

D. Golovenko. Structural and functional studies of restriction endonucleases EcoRII, BfiI and Bse634I.

G. Gasiūnas. Mechanism of DNA interference by Type II CRISPR/Cas systems.

CONFERENCES AND SEMINARS ORGANIZED IN 2012

Protein repeats: from sequence to structure and function

The Hsp90 molecular chaperone machine in yeast and mice, and in silico

Epigenomics and Complex Disease

Splicing and dicing in adenovirus-infected cells – for good or for worse

Why is Taq DNA Polymerase so Stable?

Protein Quality Control and Calcium Signalling

Single cell genomics and epigenomics

Biochemical and immunological properties of surface/secreted proteins of Staphylococcus aureus and their use as potential components for a multivalent vaccine

Magnetic tweezers: From single enzyme dynamics to mechanics of DNA nanostructures

Dynamic Protein Interactions Regulate the Cellular Response to DNA Damage

DEPARTMENT OF PROTEIN-NUCLEIC ACIDS INTERACTION

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 260 2108
E-mail:
http://www.ibt.lt/en/laboratories/department-of-protein---dna-interactions

Head - Prof. Dr. Virginijus Šikšnys

STAFF

Chief research fellows: Prof. Dr. V. Šikšnys, Dr. S. Gražulis, Dr. G. Sasnauskas.
Senior research fellows: Dr. G. Tamulaitienė, Dr. G. Tamulaitis, Dr. M. Zaremba.
Research fellows: Dr. E. Manakova, Dr. A. Šilanskas, Dr. R. Sukackaitė.  
Junior research fellows: Dr. G. Gasiūnas, Dr. D. Golovenko.
Post doctoral student: Dr. L. Jakutytė.
Doctoral students: T. Karvelis, G. Kostiuk, T. Šinkūnas, M. Ukanis.

RESEARCH PROJECTS CARRIED OUT IN 2012

Projects Supported by University Budget

Studies on Structure and Function Relationship of Enzymes and their Complexes. Prof. Dr. V. Šikšnys. 2011–2012.

The overall research theme in the Department of the Protein-Nucleic Acids Interactions is the structural and functional characterization of enzymes and enzyme assemblies that contribute to the bacteria defence systems which target invading nucleic acids. In particularly, we are involved in the in deciphering structural and molecular mechanisms of restriction enzymes, and the molecular machinery involved in the CRISPR function. We are using X-ray crystallography, mutagenesis, and functional biochemical and biophysical assays to gain information on these systems.
Type II restriction endonucleases (REases) exist in multiple oligomeric forms. The tetrameric REases have two DNA binding interfaces and must synapse two recognition sites to achieve cleavage. It was hypothesised that binding of two recognition sites by tetrameric enzymes contributes to their fidelity. We experimentally determined the fidelity for Bse634I REase in different oligomeric states. Surprisingly, we find that tetramerisation does not increase REase fidelity in comparison to the dimeric variant. Instead, an inherent ability to act concertedly at two sites provides tetrameric REase with a safety-catch to prevent host DNA cleavage if a single unmodified site becomes available.
Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both in vivo and in vitro for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. We developed two strategies to generate restriction endonuclease-TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI-TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease-TFO conjugate technology one step closer to potential in vivo applications.

Projects Supported by the Research Council of Lithuania

Structural Studies of Protein-Nucleic Acids Complexes in Solution. Dr. E. Manakova. 2011–2012.

Bacteriopage T4 Primosome. Dr. G. Tamulaitienė. 2011–2012.

Structure and Function of 5-Methyl and 5-Hydroxymethylcytosine-Directed Restriction Endonucleases. Dr. G. Sasnauskas. 2012–2014.

Function of a Molecular Motor in Atypical Restriction-Modification System. Dr. M. Zaremba. 2012–2014.

Projects of the European Social Fund under Global Grant Measure

Structure and Molecular Mechanisms of Bacterial Antivirus Defence Systems. Prof. Dr. V. Šikšnys. 2011–2015.

Contracts National and International

Studies on in Vivo Programmable Meganuclease Cas9 Functional Activity. UAB Fermentas/Thermo Fisher Scientific. Prof. Dr. V. Šikšnys. 2012.

International Grants

FP7: Strengthening and Sustaining the European Perspectives of Molecular Biotechnology in Lithuania (MoBiLi). 2009–2013.

MAIN PUBLICATIONS

Articles

G. Gasiūnas, R. Barrangou, P. Horvath, V. Šikšnys. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria // PNAS : Proceedings of the National Academy of Sciences. Washington : National Academy of Sciences. 2012, vol. vol. 109 no. 39, p. E2579-E2586.

A. Šilanskas, M. Zaremba, G. Sasnauskas, V. Šikšnys. Catalytic Activity Control of Restriction Endonuclease-Triplex Forming Oligonucleotide Conjugates // Bioconjugate chemistry. Washington : American Chemical Society. 2012, vol. 23, iss. 2, p. 203-211.

S. Gražulis, A. Daškevič, A. Merkys, D. Chateigner, L. Lutterotti, M. Quiros, N. Serebryanaya, P. Moeck, R. Downs, A. Bail. Crystallography Open Database (COD): an open-access collection of crystal structures and platform for world-wide collaboration // Nucleic Acids Research. Oxford : Oxford University Press. 2012, vol. 40, iss. D1, p. D420-D427.

E. Čapkauskaitė, A. Zubrienė, L. Baranauskienė, G. Tamulaitienė, E. Manakova, V. Kairys, S. Gražulis, S. Tumkevičius, D. Matulis. Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human carbonic anhydrases // European Journal of Medicinal Chemistry. Paris : Elsevier Masson. 2012, Vol. 51, p. 259-270.

A. Baranauskas, S. Paliksa, G. Alzbutas, M. Vaitkevičius, J. Lubienė, V. Letukienė, S. Burinskas, G. Sasnauskas, R. Skirgaila. Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants // Protein Engineering Design and Selection. Oxford : Oxford University Press. 2012, Vol. 25, no. 10, p. 657-668.

M. Ukanis, R. Šapranauskas, A. Lubys. Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency // Nucleic Acids Research. Oxford : Oxford University Press. 2012, Vol. 40, iss. 19, p. e149 (1-13).

E. Manakova, S. Gražulis, M. Zaremba, G. Tamulaitienė, D. Golovenko, V. Šikšnys. Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease // Nucleic Acids Research. Oxford : Oxford University Press. 2012, Vol. 40, no 14, p. 6741-6751.

A. Zaremba, G. Sasnauskas, V. Šikšnys. The link between restriction endonuclease fidelity and oligomeric state:a study with Bse634I // FEBS letters. Amsterdam : Elsevier BV. 2012, vol. 586, no 19, p. 3324-3329.

R. Sukackaitė, S. Gražulis, G. Tamulaitis, V. Šikšnys. The recognition domain of the methyl-specific endonuclease McrBC flips out 5-methylcytosine // Nucleic Acids Research. Oxford : Oxford University Press. 2012, Vol. 40, no 15, p. 7552-7562.

COOPERATION

Bristol University (UK)
DANISCO (France)

OTHER SCIENTIFIC ACTIVITIES

Prof. V. Šikšnys –

DEPARTMENT OF BIOLOGICAL DNA MODIFICATION

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 260 2114,
E-mail:
http://www.ibt.lt/en/laboratories/laboratory-of-biological-dna-modification

Head - Prof. Dr. Habil. Saulius Klimašauskas

STAFF

Chief research fellow: Prof. Habil. Dr. S. Klimašauskas.
Senior research fellows: Dr. R. Rakauskaitė, Dr. G. Vilkaitis, Dr. E. Kriukienė, Dr. V. Masevičius.
Research fellows: Dr. D. Daujotytė, Dr. G. Lukinavičius, Dr. Z. Liutkevičiūtė.
Junior research fellows: Dr. R. Gerasimaitė, Z. Staševskij, M. Tomkuvienė, G. Urbanavičiūtė.
Doctoral students: S. Jachimovičiūtė, A. Osipenko, S. Butkytė.

RESEARCH PROJECTS CARRIED OUT IN 2012

Projects Supported by University Budget

Nucleic Acids Modification Enzymes: Structure, Mechanisms of Action and Directed Engineering. Prof. Habil. Dr. S. Klimašauskas. 2011–2015.

The 5-methylcytosine (5-mC) derivative 5-hydroxymethylcytosine (5-hmC) is abundant in the brain for unknown reasons. In collaboration with several international research groups, we characterized the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC by using glucosylation coupled with restriction-enzyme digestion and microarray analysis. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary in human and mouse. This boundary change was mainly due to 5-hmC in the brain but due to 5-mC in non-neural contexts. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC relative to alternatively spliced exons. Our study suggests a new role for 5-hmC in RNA splicing and synaptic function in the brain.

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labelling of DNA.

Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of
spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. The described mTAG approach for the first time permits synthetically tuneable sequence-specific click-labelling of RNA with single-nucleotide precision.

The HEN1 methyltransferase from Arabidopsis thaliana modifies the 3'-terminal nucleotide of small regulatory RNAs. To better understand substrate interactions and contributions of individual steps during catalysis by HEN1, we studied binding and methylation kinetics using a series of unmethylated, hemimethylated and doubly methylated substrates. Our studies indicate that HEN1 specifically binds double-stranded unmethylated or hemimethylated miR173/miR173* substrates with sub-nanomolar affinity in a cofactor-dependent manner. Kinetic studies under single turnover and pre-steady state conditions in combination with isotope partitioning analysis showed that binary the HEN1•miRNA/miRNA* complex is catalytically competent, however successive methylation of the two strands in a RNA duplex occurs in a non-processive (distributive) manner. We also find that the observed moderate methylation strand preference is fairly independent of the nature of the 3’-terminal nucleobase and is exerted at the RNA binding step. Our results thus provide first insights into the mechanism of RNA recognition and modification by a representative small RNA 2’-O-methyltransferase.

Projects Supported by the Research Council of Lithuania

A Universal Method for Recombinant Synthesis of Selenoproteins. Dr. R. Rakauskaitė. 2012–2014.

Studies on the Biogenesis Molecular Mechanism of Non Coding RNAs in Plants. Dr. G. Vilkaitis. 2012–2014.

Projects of the European Social Fund under Global Grant Measure

Molecular Tools for Epigenomics and Rnomics. Prof. Habil. Dr. S. Klimašauskas. 2011-2015.

Contracts National and International

Analysis of DNA Modification Using DNA Methyltransferases- Mediated DNA Priming. UAB Fermentas/Thermo Fisher Scientific. Prof. Habil. Dr. S. Klimašauskas.

International Grants

NIH: Approaches for Genomic Mapping of 5-hydroxymethylcytosine a Novel Epigenetic Mark in Mammalian DNA. Prof. Habil Dr. S. Klimašauskas. 2010–2012.

FP7: Strengthening and Sustaining the European Perspectives of Molecular Biotechnology in Lithuania (MoBiLi). 2009–2013.

MAIN PUBLICATIONS

Articles

G. Lukinavičius, A. Lapinaitė, G. Urbanavičiūtė, S. Klimašauskas. Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA // Nucleic Acids Research. Oxford : Oxford University Press. 2012, Vol. 40, no 22, p. 11594-11602.

M. Tomkuvienė, B. Clouet-d'Orval, I. Černiauskas, E. Weinhold, S. Klimašauskas. Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases // Nucleic Acids Research. Oxford : Oxford University Press. 2012, Vol. 40, no 14, p. 6765-6773.

R. Sakaguchi, A. Giessing, Q. Dai, G. Lahoud, Z. Liutkevičiūtė, S. Klimašauskas, J. Piccirilli, F. Kirpekar, Y. Hou. Recognition of guanosine by dissimilar tRNA methyltransferases // RNA. New York : Cold Spring Harbor Laboratory Press. 2012, Vol. 18, iss. 9, p. 1687-1701.

E. Kriukienė, Z. Liutkevičiūtė, S. Klimašauskas. 5-Hydroxymethylcytosine - the elusive epigenetic mark in mammalian DNA // Chemical Society Reviews. Cambridge : RSC Publications. 2012, vol 41, no 21, p. 6916-6930.

T. Khare, S. Pai, K. Koncevičius, M. Pal, E. Kriukienė, Z. Liutkevičiūtė, M. Irimia, P. Jia, C. Ptak, M. Xia, R. Tice, M. Tochigi, S. Morera, A. Nazarians, D. Belsham, A. Wong, B. Blencowe, S. Wang, P. Kapranov, R. Kustra, V. Labrie, S. Klimašauskas, A. Petronis. 5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary // Nature Structural and Molecular Biology. New York : Nature Publishing Group. 2012, Vol. 19, iss. 10, p. 1037-1043.

COOPERATION

University of Paul Sabatier (France)
Institute of Biochemistry and Cell Biology (Republic of China)
Institute for Organic Chemistry (Germany)
Centre for Addiction and Mental Health, University of Toronto (Canada)
International Institute of Cellular and Molecular Biology (Poland)
Institute of Molecular Genetics (Russia)
Moscow State University (Russia)
Catholic University of Leuven (Belgium)
Thomas Jefferson University Philadelphia (US)
Institute of Systems Biology and Bioinformatics (Taiwan)

OTHER SCIENTIFIC ACTIVITIES

Prof. Habil. Dr. S. Klimašauskas

DEPARTMENT OF EUKARYOTE GENETIC ENGINEERING

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 260 2104
E-mail:
 http://www.ibt.lt/en/laboratories/egil_en

Head – Doc. Dr. Gintautas Žvirblis

STAFF

Chief research fellows: Prof. Habil. Dr. K. Sasnauskas, Habil. Dr. I. Meškienė, Dr. A. Gedvilaitė.
Senior research fellows: Dr. E. Mištinienė, Dr. L. Mažutis, Dr. A. Ražanskienė, Dr. R. Slibinskas, Dr. G. Žvirblis.
Research fellows: Dr. A. Abraitienė, Dr. R. Abraitis, Dr. L. Antoniukas, Dr. V. Kazanavičiūtė, Dr. R. Petraitytė-Burneikienė.
Junior research fellows: Dr. A. Bulavaitė, Dr. E. Čiplys, Dr. M. Juozapaitis, Dr. R. Ražanskas, Dr. M. Zaveckas.
Doctoral students: G. Alzbutas, E. Biveinytė, M. Norkienė, T. Povilaitis, P. L. Tamošiūnas, R. Vorobjovienė, J. Rutkauskaitė, R. Galinis, K. Kvederavičiūtė, J. Lazutka, G. Žvirblytė.

RESEARCH PROJECTS CARRIED OUT IN 2012

Projects Supported by University Budget

Synthesis of Chimeric and Native Virus Proteins in Yeast and their Application. Prof. K. Sasnauskas, Dr. G. Žvirblis. 2012.

Research direction for analysis of chimeric and native viral proteins in yeast and their practical application was intensively exploited in 2012. Virus-like particles expressed in yeast allowed analysis of serological cross-reactions between four polyomaviruses of the wild birds. Recombinant pseudo type virus-like particles harbouring inserted target antigen generated antibodies against cellular marker p16INK4A. Hantavirus proteins obtained in yeast were used for infection analysis of wild small mammals in South India. Yeast expressed recombinant viral proteins have been used for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus. The data of codon influence on heterologous production of human papillomavirus type 16 major structural protein L1 in yeast have been evaluated. Induction of cytosolic unfolded proteins in Saccharomyces cerevisiae wasshowed during over-expression of human virus surface glycoprotein precursors. Improvement of measles surface glycoprotein solubility has been demonstrated during co-expression with human calnexin in yeast.

Projects Supported by Research Council of Lithuania

Analysis and Diagnostics of New Human Parvoviridea Family Viruses. Prof. Habil. Dr. K. Sasnauskas. 2011–2012.

Regulation of Signal Transduction in A.Thialiana. Habil. Dr. I. Meškienė. 2011–2012.

Stress Response Analysis on the Proteome Level in Cells Producing Recombinant Proteins. Dr. R. Slibinskas. 2011–2012.

Influence on Viroid and Plant Host Interaction of High Ozone Concentration. Dr. A. Abraitienė. 2011–2012.

High-Throughput Screening of Antibody-Secreting Cells Using Droplet-Based Microfluidics. Dr. L. Mažutis. 2012–2014.

Interspecific Hybrids of Orchard Plant - a Novel Source of Anthocyanins. Dr. Ražanskas. 2011–2014.

Expression Analysis of Anthocyanin Biosynthesis Genes in Horticultural Plants. Dr. V. Kazanavičiūtė. 2012–2015.

Projects Supported by Agency for Science, Innovation And Technology

Development of Microfluidics Technology for Monodisperse Vesicles Production and Improved Drug Delivery. Dr. L. Mažutis. 2012–2013.

Projects of the European Social Fund under Global Grant Measure

The Use of Genome-Wide Analysis for Engineering of New Yeast Strains with Improved Heterologous Expression. 2012–2015. Dr. R. Slibinskas.

National Integrated Program

Biotechnology and Biopharmacy: Fundamental and Applied Research.
SWISS-LT
Directed Evolution of Computer Designed Enzymes Using Droplet-Based Microfluidics. Dr. L. Mažutis. 2012–2016.

Signalling Control of Pathogen Induced Plant Immunity. Habil. Dr. I. Meškienė. 2012–2016.

Contracts National and International

Recombinant Viral Proteins. Abcam Ltd, London, UK.  Dr. G. Žvirblis. 2012.

Construction of Recombinant Hantavirus Nucleocapsid Proteins. Euroimmun AG. A. Ražanskienė. 2012.

International Grants

FP7: Development of Novel Antiviral Drug against Influenza (FLUCURE). Dr. G. Žvirblis. 2010–2013.

FP7: Strengthening and Sustaining the European Perspectives of Molecular Biotechnology in Lithuania (MoBiLi). 2009–2013.

MAIN PUBLICATIONS

Articles

Y. Skhiri, P. Gruner, B. Semin, Q. Brosseau, D. Pekin, L. Mažutis, V. Goust, F. Kleinschmidt, H. El, J. Hutchison, E. Mayot, J. Bartolo, A. Griffiths, V. Taly, J. Baret. Dynamics of molecular transport by surfactants in emulsions // Soft matter. Cambridge : RSC Publications. 2012, Vol. 8, iss. 41, p. 10618-10627.

E. Mažeikė, A. Gedvilaitė, U. Blohm. Induction of insert-specific immune response in mice by hamster polyomavirus VP1 derived virus-like particles carrying LCMV GP33 CTL epitope // Virus research. Amsterdam : Elsevier BV. 2012, vol. 163, iss. 1, p. 2-10.

M. Norkienė, A. Gedvilaitė. Influence of Codon Bias on Heterologous Production of Human Papillomavirus Type 16 Major Structural Protein L1 in Yeast // The Scientific World Journal. New York : Hindawi Publishing Corporation. 2012, Vol. 2012, Art. ID 979218 (6 p.).

M. Schlegel, E. Tegshduuren, K. Yoshimatsu, R. Petraitytė, K. Sasnauskas, B. Hammerschmidt, R. Friedrich, M. Mertens, M. Groschup, S. Arai, R. Endo, K. Shimizu, T. Koma, S. Yasuda, C. Ishihara, R. Ulrich, J. Arikawa, B. Kollner. Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus // Archives of Virology. Wien : Springer. 2012, Vol. 157, p. 2179-2187.

L. Mažutis, A. Griffiths. Selective droplet coalescence using microfluidic systems // Lab On a Chip: miniaturisation for chemistry, physics, biology and bioengineering. Cambridge : Royal Society of Chemistry. 2012, Vol. 12, iss. 10, p. 1800-1806.

A. Zielionka, A. Gedvilaitė, J. Reetz, U. Rosler, H. Muler, R. Johne. Serological cross-reactions between four polyomaviruses of birds using virus-like particles expressed in yeast // Journal of general virology. Reading : Society for General Microbiology. 2012, vol. 93, p. 2658-2667.

R. Lasickienė, A. Gedvilaitė, M. Norkienė, V. Simanavičienė, I. Šėžaitė, D. Dekaminavičiūtė, E. Shikova, A. Žvirblienė. The Use of Recombinant Pseudotype Virus-Like Particles Harbouring Inserted Target Antigen to Generate Antibodies against Cellular Marker p16INK4A // The Scientific World Journal. New York : Hindawi Publishing Corporation. 2012, Vol. 2012, Art. ID 263737 (8 p.).

COOPERATION

Institute for Novel and Emerging Infectious Diseases (Germany)
Institute of Experimental Pathology and Parasitology (Bulgaria)
Friedrich-Loeffler-Institut Bundesforschungsinstitut für Tiergesundheit, Federal Research Institute for Animal Health OIE Collaborating Centre for Zoonoses in Europe (Germany)
Max Planck Institute for Molecular Plant Physiology (Germany)
Technische Universität München (Germany)
Department of Virology, University of Freiburg (Germany)

OTHER SCIENTIFIC ACTIVITIES

Habil. Dr. I. Meškienė –

DEPARTMENT OF IMMUNOLOGY AND CELL BIOLOGY

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 260 2117
E-mail:
http://www.ibt.lt/en/laboratories/ilbl_en

Head – Prof. Dr. Aurelija Žvirblienė

STAFF

Chief research fellow: Dr. A. Žvirblienė.
Senior research fellows: Dr. A. Kanopka, Dr. S. Laurinavičius, Dr. J. Matulienė, Dr. M. Plečkaitytė, Dr. P. Stakėnas.
Research fellow: Dr. I. Kučinskaitė-Kodzė.
Junior research fellows: D. Bakonytė, E. Jakubauskienė, R. Lasickienė.
Doctoral students: V. Simanavičienė, D. Dekaminavčiūtė, I. Dalgėdienė.

RESEARCH PROJECTS CARRIED OUT IN 2012

Projects Supported by University Budget

Development of Monoclonal and Recombinant Antibodies. Dr. A. Žvirblienė. 2011–2015.

Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce high-affinity scFv-Fc proteins in yeast expression system.  The scFv protein derived from selected hybridoma cell line was fused with human IgG1 Fc domain. Two different variants of scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. It was demonstrated that the fusion of scFv-Fc molecules with hamster polyomavirus -derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong antigen-binding activity.
This approach allowed generation of multivalent scFv-Fc proteins displayed on the surface of pseudotype VLPs. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. In conclusion, the current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins

Research on Molecular Epidemiology of Tuberculosis. Dr. P. Stakėnas. 2011–2015.

Tuberculosis which is caused by M. tuberculosis complex bacteria remains a serious health problem in Lithuania. The rate of incidence, particularly multidrug-resistant tuberculosis is one of the highest in the European Community. The aims of this project are to characterize in detail population of infectious agent circulating in Lithuania by the means of molecular epidemiology and to clarify the genetic determinants leading to the emergence of drug resistance. The research is carried out in close collaboration with Infectious Diseases and Tuberculosis Hospital, affiliate of the public institution Vilnius University Hospital Santariškių Klinikos. In 2012, we focussed on spoligotyping and 24-locus MIRU-VNTR typing of new clinical strains and identification of MIRU-VNTR patterns in kanamycin-resistant population of M. tuberculosis. The results demonstrated an ongoing transmission of specific strains and were in agreement with a speculation that the mutations causing kanamycin resistance could be an important driving force for the emergence and spread of extensively drug-resistant strains in Lithuania. In the field of mutations we continued search for the novel targets involved in M. tuberculosis resistance to the reserve aminoglycosides and capreomycin and characterization spectrum of the mutations in the genes (rrs, rpsL, gidB) implicated in resistance to streptomycin.

Influence of Mutations in CBL Gene for Pre-Mrna Splicing in Case of Juvenile Myelomonocytic Leukemia. Dr. A. Kanopka. 2011–2012.

Cbl (Casitas B-lineage Lymphoma) is a mammalian gene encoding several proteins including E3 ubiquitin-protein ligase CBL involved in cell signalling and protein ubiquitination. Missense mutationsin Cbl gene have recently been associated with juvenile myelomonocyticleukaemia (JMML), an aggressive myeloproliferative and myelodysplastic neoplasm of early childhood characterized by excessive macrophage/monocyte proliferation. CBL protein controls proliferative signalling networks by down-regulating the growth factor receptor signalling cascades in various cell types. The aim of the project is to determine mutation influence in Cbl gene for mRNA processing at the level of pre-mRNA splicing.
Our studies revealed that mutations in intron sequences close to 3’ splice site in Cbl pre-mRNA change mRNA processing by affecting splice site usage. Our data also showed that interaction of 60-70 kDa RNA binding proteins with mutated pre-mRNA is much weaker. We identify that one of the differential interacting protein is the essential splicing factor U2AF65.

Projects Supported by Research Council of Lithuania

Studies on the Mechanism of the Cytolytic Activity of the Bacterial Toxin Vaginolysin. Dr. M. Plečkaitytė. 2012–2014.

Development of Recombinant Antibodies against Carbonic Anhydrase. Dr. A. Žvirblienė. 2012–-2014.

Influence of Alternative Pre- Irna Splicing on Lytic Replication of Kaposi Sarcoma Herpes Virus. Dr. S. Laurinavičius. 2012–2014.

Studies on Allergic Diseases Genetic and Environmental Risk Factors in the Lithuanian Newborn Cohort. Dr. A. Žvirblienė. 2012–2014.

Molecular Mechanisms in Alzheimer's Disease. Dr. A. Žvirblienė. 2012–2014.

Splicing Factors and their Regulated Mirna as Cancer Biomarkers for Gastrointestinal System.  Dr. A. Kanopka. 2012–2014.

Projects supported by Agency for Science, Innovation and Technology

Metastatic Tumours Facilitated by Hypoxic Tumour Micro-Environments (METOXIA). Dr. A. Kanopka.

Pan-European Network for the Study and Clinical Management of Drug Resistant Tuberculosis (TB PAN-NET). Dr. P. Stakėnas.

Contracts National and International

Generation of Monoclonal Antibodies. Santa Cruz Biotechnology. Inc. US. Dr. A. Žvirblienė.

Generation of Monoclonal Antibodies. Abcam Ltd, London, UK Dr. A. Žvirblienė.

Projects of the European Social Fund under Global Grant Measure

Novel Chimeric Proteins with Antiviral Activity. Dr. A. Žvirblienė. 2012–2015.

International Grants

FP7: Metastatic Tumours Facilitated by Hypoxic Tumour Micro-Environments (METOXIA). Dr. A. Kanopka. 2009–2013.

FP7:  Pan-European Network for the Study and Clinical Management of Drug Resistant Tuberculosis (TB PAN-NET). Dr. P. Stakėnas. 2008–2013.

FP7: Strengthening and Sustaining the European Perspectives of Molecular Biotechnology in Lithuania (MoBiLi). VU Institute of Biotechnology. 2009–2013.

MAIN PUBLICATIONS

Articles

B. de, K. Kremer, C. Kodmon, P. Supply, S. van, P. Stakėnas. First worldwide proficiency study on variable-number tandem-repeat typing of mycobacterium tuberculosis complex strains / Jessica L. de Beer, Kristin Kremer, Csaba Kodmon, Philip Supply, Dick van Soolingen, and the Global Network for the Molecular Surveillance of Tuberculosis 2009 [from LithuaniaPetras Stakėnas] // Journal of clinical microbiology. Washington : American Society for Microbiology. 2012, vol. 50, no 3, p. 662-669.

E. Jakubauskienė, V. Janavičiūtė, I. Pečiulienė, P. Soderkvist, A. Kanopka. G/A polymorphism in intronic sequence affects the processing of MAO-B gene in patients with Parkinson disease // FEBS letters. Amsterdam : Elsevier BV. 2012, Vol. 586, iss. 20, p. 3698-3704.

M. Plečkaitytė, M. Janulaitienė, R. Lasickienė, A. Žvirblienė. Genetic and biochemical diversity of Gardnerella vaginalis strains isolated from women with bacterial vaginosis // FEMS Immunology and Medical Microbiology. Oxford : Wiley-Blackwell Publishing Ltd. 2012, Vol. 65, iss. 1, p. 69-77.

M. Plečkaitytė, M. Zilnytė, A. Žvirblienė. Insights into the CRISPR/Cas system of Gardnerella vaginalis // BMC microbiology. London : BioMed Central Ltd. 2012, vol. 12, p. art. no 301.

R. Dubakienė, O. Rudzevičienė, I. Būtienė, I. Šėžaitė, M. Petronytė, D. Vaicekauskaitė, A. Žvirblienė. Studies on early allergic sensitization in the Lithuanian birth cohort // The Scientific World Journal. New York : Hindawi Publishing Corporation. 2012, Vol. 2012, Art. ID 909524 (6 p.).

R. Lasickienė, A. Gedvilaitė, M. Norkienė, V. Simanavičienė, I. Šėžaitė, D. Dekaminavičiūtė, E. Shikova, A. Žvirblienė. The Use of Recombinant Pseudotype Virus-Like Particles Harbouring Inserted Target Antigen to Generate Antibodies against Cellular Marker p16INK4A // The Scientific World Journal. New York : Hindawi Publishing Corporation. 2012, Vol. 2012, Art. ID 263737 (8 p.).

E. Kazlauskas, V. Petrikaitė, V. Michailovienė, J. Revuckienė, J. Matulienė, L. Grinius, D. Matulis. Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90 // PLoS ONE [Elektroninis išteklius]. San Francisko : Public Library of Science. 2012, vol. 7, iss. 5, p. e36899 (12 p.).

I. Devaux, D. Manissero, l. de, K. Kremer, S. van, A. Sosnovskaja, P. Stakėnas. Surveillance of extensively drug-resistant tuberculosis in Europe, 2003-2007 / I. Devaux, D. Manissero, K. Fernandez de la Hoz, K. Kremer, D. van Soolingen, on behalf of the EuroTB network [from Lithuania A. Sosnovskaja, P, Stakėnas] // Eurosurveillance. Saint-Maurice : Centre Europeen pour la Surveillance Epidemiologique du SIDA. 2010 vol. 15, no 18, p. [1-6].

Patents

D. Matulis, I. Čikotienė, E. Kazlauskas, J. Matulienė. 5-aryl-4-(5-substituted 2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of HSP90 chaperone and the intermediates for production thereof // 20120201.

D. Matulis, I. Čikotienė, E. Kazlauskas, J. Matulienė. 5-aryl-4-(5-substituted 2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chaperone and the intermediates for production thereof : patent no.: US 8,314,132 B2 / inventors: Matulis; Daumantas (Vilnius, LT), Cikotiene; Inga (Vilnius, LT), Kazlauskas; Egidijus (Vilnius, LT), Matuliene; Jurgita (Vilnius, LT) // 20121120.

COOPERATION

Karolinska Institute (Sweden)
Tsukuba University (Japan)
Oslo University (Norway)
Emerging Bacterial Pathogens Unit (Holland)
Wurzburg University (Germany) 
Friedrich-Loeffler-Institute (Germany
Institute of Experimental Morphology, Pathology and Anthropology with Museum of the Bulgarian Academy of Sciences (Bulgaria)

OTHER SCIENTIFIC ACTIVITIES

DEPARTMENT OF BIOTHERMODYNAMICS AND DRUG DESIGN

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 269 1884
E-mail:
http://www.ibt.lt/en/laboratories/laboratory-of-biothermodynamics-and-drug-design

Head – Dr. Daumantas Matulis

STAFF

Chief research fellow: Dr. D. Matulis.
Senior research fellows: Dr. V. Petrauskas, Dr. V. Smirnovas.
Research fellows: Dr. V. Dudutienė, Dr. V. Petrikaitė, Dr. A. Zubrienė.
Junior research fellows: J. Jachno, V. Michailovienė.
Doctoral students
: L. Baranauskienė, D. Timm, V. Raškauskas, D. Daniel Timm, V. Raškauskas.  

RESEARCH PROJECTS CARRIED OUT IN 2012

Projects Supported by University Budget

Screening of Active Compounds by Structural Biothermodynamics. Dr. D. Matulis. 2011–2012.

1. Design of selective inhibitors for human carbonic anhydrase (CA) isozymes as anticancer targets
Numerous human recombinant CAs were produced at DBDD. Antibodies against hCA XII were further developed and used as markers for certain cancers with our collaborators at Tampere University. An RT-PCR method was designed to quantify hCA XII expression. A large group of inhibitors were synthesized or obtained from collaborators at the Institute of Organic Synthesis in Latvia. The most potent inhibitors exhibited affinities in the single-digit nanomolar range as determined by biophysical techniques, isothermal titration calorimetry and the thermal shift assay. One group of inhibitors was published in the European Journal of Medicinal Chemistry. Several groups of inhibitors could be selected for continued development as drug leads.
2. Thermodynamics of inhibitor binding to human Hsp90 chaperone
Thermodynamic and structural characterization of ICPD lead compound series was finished in 2011 and the results were published in two publications, one in collaboration with British scientists. The European and USA patent for the ICPD compound series has been granted.

Projects Supported by the Research Council of Lithuania

Synthesis of Fluorine Benzimidazole Sulfonamides and Analysis of their Interaction with Carboanhydrases. Dr. V. Dudutienė. 2011–2012

Looking for the Origins of Mammalian Prion 'Strains'. Dr. V. Smirrnovas. 2012–2014.

Carbonic Anhidrase hCA XII as a Potential Marker for Cancer Cells. Dr. D. Matulis. 2012–2014.

Projects of the European Social Fund under Global Grant Measure

Exploring Flavones as Universal Inhibitors of Amyloid-like Fibril Formation. Dr. V. Smirnovas. 2012–2015.

Design of Selective Carbonic Anhydrase, Hsp90, and Hsp70 Inhibitors and Investigation of their Anticancer Properties. Dr. D. Matulis. 2012–2015.

International Grants

FP7: Towards Construction of a Comprehensive Map of Amyloid-Ligand Interactions:(-)-Epigallocatechin 3-Gallate and Insulin Amyloid. (EGCG+INSULIN=). Dr. V. Smirnovas. 2011–2015.

FP7: Strengthening and Sustaining the European Perspectives of Molecular Biotechnology in Lithuania (MoBiLi). VU Institute of Biotechnology. 2009–2013.

MAIN PUBLICATIONS

Articles

S. Sharp, S. Roe, E. Kazlauskas, I. Čikotienė, P. Workman, D. Matulis, C. Prodromou. Co-Crystalization and In Vitro Biological Characterization of 5-Aryl-4-(5-Substituted-2-4-Dihydroxyphenyl)-1,2,3-Thiadiazole Hsp90 Inhibitors // PLoS ONE [Elektroninis išteklius]. San Francisko : Public Library of Science. 2012, Vol. 7, iss. 9, p. e44642 (8 p.).

E. Čapkauskaitė, A. Zubrienė, L. Baranauskienė, G. Tamulaitienė, E. Manakova, V. Kairys, S. Gražulis, S. Tumkevičius, D. Matulis. Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human carbonic anhydrases // European Journal of Medicinal Chemistry. Paris : Elsevier Masson. 2012, Vol. 51, p. 259-270.

L. Pirrie, A. McCarthy, L. Major, V. Morkūnaitė, A. Zubrienė, D. Matulis, S. Lain, T. Lebl, N. westwood. Discovery and validation of SIRT2 inhibitors based on tenovin-6: use of a 1H-NMR method to assess deacetylase activity // Molecules. Basel: MDPIAG. 2012, vol. 17, no 10, p. 12206-12224.

B. Giessrigl, S. Krieger, M. Rosner, N. Huttary, P. Saiko, M. Alami, S. Messaoudi, J. Peyrat, A. Maciuk, M. Gollinger, S. Kopf, E. Kazlauskas, P. Mazal, T. Szekeres, M. Hengstschlager, D. Matulis, W. Jager, G. Krupitza. Hsp90 stabilizes Cdc25A and counteracts heat shock-mediated Cdc25A degradation and cell-cycle attenuation in pancreatic carcinoma cells // Human molecular genetics. Oxford : Oxford University Press. 2012, Vol. 21, no. 21, p. 4615-4627.

L. Baranauskienė, D. Matulis. Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII // BMC Biophysics. London : BioMed Central Ltd. 2012, Vol. 5, art. no. 12 (21 p.).

Z. Toleikis, P. Cimmperman, V. Petrauskas, D. Matulis. Serum albumin ligand binding volumes using high pressure denaturation // The journal of chemical thermodynamics. London : Academic Press, p. 24-29.

L. Labanauskas, V. Dudutienė, G. Urbelis, J. Šarlauskas, J. Sūdžius, D. Matulis, R. Striela, A. Žilinskas. Synthesis of substituted 2Ī»4Ī´2-[1,2,3]thiadiazolo[3,4-c]benzimidazoles and 2Ī»4Ī´2-[1,2,3,5]thiatriazolo[3,4-c]benzimidazoles // ARKIVOC : Archive for Organic Chemistry. Gainesville : ARKAT USA, Inc. 2012, part. 8, p. 17-26.

E. Kazlauskas, V. Petrikaitė, V. Michailovienė, J. Revuckienė, J. Matulienė, L. Grinius, D. Matulis. Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90 // PLoS ONE [Elektroninis išteklius]. San Francisko : Public Library of Science. 2012, vol. 7, iss. 5, p. e36899 (12 p.).

P. Norvaišas, V. Petrauskas, D. Matulis. Thermodynamics of Cationic and Anionic Surfactant Interaction // The journal of physical chemistry B. Washington : American Chemical Society. 2012, vol. 116, iss. 7, p. 2138-2144.

E. Čapkauskaitė, S. Tumkevičius, D. Matulis. Synthesis of benzenesulfonamides containing azaheterocyclic moieties // Latvian Journal of Chemistry. Salaspils : Institute of Inorganic Chemistry of the Riga Technical University. 2012, no. 1 : Materials of the Paul Walden 7th Symposium on organic chemistry (Riga, September 12-13, 2011), p. 54.

Patents

D. Matulis, I. Čikotienė, E. Kazlauskas, J. Matulienė. 5-aryl-4-(5-substituted 2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of HSP90 chaperone and the intermediates for production thereof // 20120201.

D. Matulis, I. Čikotienė, E. Kazlauskas, J. Matulienė. 5-aryl-4-(5-substituted 2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chaperone and the intermediates for production thereof : patent no.: US 8,314,132 B2 / inventors: Matulis; Daumantas (Vilnius, LT), Cikotiene; Inga (Vilnius, LT), Kazlauskas; Egidijus (Vilnius, LT), Matuliene; Jurgita (Vilnius, LT) // 20121120.

COOPERATION

Institute of Biochemistry, Vilnius University (Lithuania)
Institute of Oncology, Vilnius University (Lithuania)
Centre for Innovative Medicine (Lithuania)
Institute of Medical Technology, University of Tampere (Finland)
International Institute of Molecular and Cell Biology (Poland)
Polish Medical Research Centre, Polish Academy of Sciences, Dept.Exp. Pharmacology (Poland)
Lead Generation Biology at Johnson & Johnson Pharmaceutical Research and Development (USA)
Centre for Structural Biochemistry (France)
Cancer Research Centre, University of Edinburgh (UK)
University of Tubingen (Germany)
TA Instruments (Sweden)
Genome Damage and Stability Centre, University of Sussex (UK)
Latvian Institute of Organic Synthesis (Latvia)
University of Florence (Italy)
Linköping University (Sweden)
JSC Amilina (Lithuania)

OTHER SCIENTIFIC ACTIVITIES

Dr. D. Matulis –

  • member of the international organizing committee  of the 1st Central and Eastern European  conference on Thermal Analysis and Calorimetry, Craiova (Romania), http://www.ceec-tac.org/comittees.html;
  • editorial board  member of the international journal BMC Biophysics.

SECTOR OF APPLIED BIOCATALYSIS

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 240 4679
E-mail:

Head – Dr. Inga Matijošytė

STAFF

Research fellow: Dr. I. Matijošytė.
Junior research fellows: Dr. R. Gruškienė, Dr. M. B. Meizeraitytė, Dr. B. Pudžiuvytė, R. Šiekštelė.
Doctoral students: M. Šulcienė, V. Matikevičienė, I. Gailiūtė.

RESEARCH PROJECTS CARRIED OUT IN 2012

Projects Supported by University Budget

Development and Investigation of Novel Biocatalysts and their Respective Processes. Dr. I. Matijošytė. 2011–2012.

Sector of Applied Biocatalysis (SAB) seeks to develop biocatalysts with novel activities and/or biocatalytical processes by the three most common ways: screening for enzymes (environmental samples, enzyme and strain collections, metagenomic and expression databases), development of biocatalyst (gene engineering, development of analytical systems) and application of biocatalyst (immobilization, recycling, proof of principal, activity/selectivity, stability, reaction media).
The research for the year 2012 was performed in several directions. The metagenomic library was created from the water penetrated through rubbish dump and one clone expressing enzyme with lipase activity has been obtained. Development of new enzymes for biopolyol synthesis is in progress: enzyme with epoxidation activity was found in Bacillus pumilus bacteria isolated from sewage water which was enriched with vegetable oil. Cultivation conditions and purification scheme were established for this specific enzyme. Laccase from L. rufus and secondary alcohol oxidase from Pseudomonas putida 4 were successfully purified and have been submitted for N-terminus sequencing. In the project Development of innovative biotechnology for oil base lubricant production, BIOLUBRICANT (No. 31V-1, MITA), lipase from Serratia sp. has been isolated, purified, sequenced and cloned into the yeast K. lactis expression system. From April 2012 the project The development of innovative biocatalytic stain remover, FASTREMOVE (No. 31-38, MITA) has been started. The aim of this project is to establish a methodology for the use of enzymes in “express” stain removers.

Projects supported by Agency for Science, Innovation and Technology

The Development of Innovative Biocatalytic Stain Remover. Dr. I. Matijošytė. 2011–2013.

Development of Innovative Biotechnology for Oil Base Lubricant Production. Dr. I. Matijošytė. 2011–2013.

COOPERATION

University of Applied Sciences (Switzerland)
TU Delft (Netherlands)
University of Pizza (Italy)
Centre of Innovative Medicine (Lithuania)

DEPARTMENT OF BIOINFORMATICS

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 269 1881
E-mail:
http://www.ibt.lt/bioinformatics

Head - Dr. Česlovas Venclovas

STAFF

Chief research fellow: Dr. Č. Venclovas.
Senior research fellows: Dr. V. Kairys, Dr. I. Mitašiūnaitė-Besson, Dr. M. Margelevičius, Dr. A. Timinskas.
Post doctoral student: J. Dapkūnas.
Doctoral students
: D. Kazlauskas, J. Višinskienė, K. Timinskas, K. Olechnovič.

RESEARCH PROJECTS CARRIED OUT IN 2012

Projects Supported by University Budget

Studies on Protein Structure, Function and Evolution through Computational Methods. Dr. Č. Venclovas. 2011–2013.

In 2012 we focused on the development of methods for protein structure evaluation and on the computational characterization of proteins functioning in DNA replication, an essential process in all life forms.

Development of methods for evaluation protein structure models against the reference structure

Evaluation of protein models against the native (reference) structure is essential for the development and benchmarking of protein structure prediction methods. Although a number of evaluation scores have been proposed to date, many aspects of model assessment still lack desired robustness. To address the shortage of effective assessment methods, we developed CAD-score, a new evaluation function quantifying differences between physical contacts in a model and the reference structure. The new score uses the concept of residue-residue contact area difference (CAD). Contact areas, the underlying basis of the score, are derived using the Voronoi tessellation of protein structure. Our newly developed CAD-score has a number of attractive features. It is a continuous function, confined within fixed limits, free of any arbitrary thresholds or parameters. The built-in logic for treatment of missing residues allows consistent ranking of models of any degree of completeness. Our analysis has shown that CAD-score displays a balanced assessment of domain rearrangement, removing the necessity for different treatment of single-domain, multi-domain and multi-subunit structures. Moreover, CAD-score makes it possible to assess the accuracy of inter-domain or inter-subunit interfaces directly. In addition, our new approach offers an alternative to the superposition-based model clustering. We have implemented CAD-score both as a web server and a standalone software package available at http://www.ibt.lt/bioinformatics/cad-score/. The study has been reported in “Proteins: Structure, Function and Bioinformatics”.

Computational detection and analysis of the single-stranded DNA binding (SSB) proteins in Nucleo-Cytoplasmic Large DNA Viruses

In this study we focused on DNA replication of eukaryotic nucleo-cytoplasmic large DNA viruses (NCLDVs). This group of double-stranded (ds) DNA viruses includes the largest currently known viruses. For example, Mimivirus, a member of NCLDVs, has a genome, which is larger than that of some bacteria. At present NCLDVs are classified into seven families. Replication of these large viruses typically does not strongly depend on the host DNA replication and transcription systems. In line with the large genome size and the relative replication independence, comparative genomics studies revealed that NCLDV genomes encode essential DNA replication proteins including DNA polymerase, helicase and primase. However, all these analyses failed to detect NCLDV homologs of canonical single-stranded DNA binding (SSB) proteins. Given the importance of SSB proteins in various DNA transactions, the seeming absence of canonical SSB proteins in NCLDV genomes intrigued us.

Therefore, we set out to investigate the case of “missing” canonical SSB proteins. Using advanced computational methods, we unexpectedly detected homologs of the bacteriophage T7 SSB protein (gp2.5) present in four NCLDV families We found the properties of these homologs to be consistent with the SSB function. Moreover, we were able to implicate specific residues in ssDNA binding. Even more surprisingly, we found that previously characterized SSB proteins in poxviruses are evolutionary distinct. Instead of homology with T7 SSB we detected their evolutionary link with the bacterial RNA-binding protein SmpB. Thus, apparently, the NCLDVs have the two major distinct sets of SSB proteins having bacteriophage and bacterial origins respectively. The results of the study have been published in the Bioinformatics.

Projects Supported by Research Council of Lithuania

Structure, Classification and Distribution of Bacterial C Family DNA Polymerases. Dr. Č. Venclovas. 2011–2012.

Up to Date Method for Determination of Evolutionary Relations among Proteins CONDOR. Dr. M. Margelevičius. 2011–2012.

International Grants

FP7: Strengthening and Sustaining the European Perspectives of Molecular Biotechnology in Lithuania (MoBiLi). VU Institute of Biotechnology. 2009–2013.

MAIN PUBLICATIONS

Articles

E. Čapkauskaitė, A. Zubrienė, L. Baranauskienė, G. Tamulaitienė, E. Manakova, V. Kairys, S. Gražulis, S. Tumkevičius, D. Matulis. Design of [(2-pyrimidinylthio)acetyl]benzenesulfonamides as inhibitors of human carbonic anhydrases // European Journal of Medicinal Chemistry. Paris : Elsevier Masson. 2012, Vol. 51, p. 259-270.

A. Nouri, R. Castro, V. Kairys, J. Santos, J. Rodrigues, Y. Li, H. Tomas. Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studies // Journal of materials science: materials in medicine. New York : Springer New York LLC. 2012, vol. 23, no 12, p. 2967-2980.

L. Nagirnaja, Č. Venclovas, K. Rull, K. Jonas, H. Peltoketo, O. Christiansen, V. Kairys, G. Kivi, R. Steffensen, I. Huhtaniemi, M. Laan. Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin Ī²-subunit // Molecular Human Reproduction. Oxford : Oxford University Press. 2012, Vol. 18, no. 8, p. 379-390.

D. Kazlauskas, Č. Venclovas. Two distinct SSB protein families in nucleo-cytoplasmic large DNA viruses // Bioinformatics. Oxford : Oxford University Press. 2012, Vol. 28, no. 24, p. 3186-3190.

Č. Venclovas. Methods for Sequence-Structure Alignment // Homology Modeling: Methods and Protocols / eds.: Andrew J.W. Orry, Ruben Abagyan. Series: Methods in Molecular Biology, Vol. 857. ISSN 1064-3745. New York : Humana Press, Inc. p. 55-82.

COOPERATION

North Eastern University (USA)
University of Cape Town (South Africa)
Tel Aviv University (Israel)
Tartu University (Estonia)

DNA SEQUENCING CENTRE

8 V. A. Graičiūno, LT 02241 Vilnius
Tel. 269 1883
E-mail:
http://www.ibt.lt/sc/index.htm

Head - Eglė Rudokienė

COOPERATION

Lithuanian University of Health Sciences (Lithuania)
Nature Research Centre (Lithuania)
Vytautas Magnus University (Lithuania)