Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU
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| Опубліковано в: : | Вiopolymers and Cell |
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| Дата: | 2016 |
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Інститут молекулярної біології і генетики НАН України
2016
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| Цитувати: | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU // Вiopolymers and Cell. — 2016. — Т. 32, № 5. — С. 395-405. — англ. |
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| citation_txt | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU // Вiopolymers and Cell. — 2016. — Т. 32, № 5. — С. 395-405. — англ. |
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395
X Conference of Young Scientists
Institute of Molecular Biology and Genetics NAS of Ukraine
26-27 May 2015
Loss of alpha-e-catenin in embrionic heart leads to dramatic
malformation of adult heart
V. V. Balatskyi, O. L. Palchevska, L. L. Macewicz, O. O. Piven, L. L. Lukash
Institute of Molecular Biology and Genetics, Department of Human Genetics,
Kyiv, Ukraine
kostikov1212@gmail.com
Aim. In our work we have focused on the embryonic cardiospecific ablation of alpha-E-catenin and its reflec-
tion on heart adult heart formation. Methods. We studied the significance of embryonic cardiospecific ablation
of alpha-E-catenin for heart aging using the conditional knockout approach. We analyzed how alpha-E-catenin
haploinsufficiency and homozygotic deletion affected the postnatal heart development and adult heart forma-
tion. For this we used measurement of the heart weight/body weight (HW/BW) and heart weight/tibia length
(HW/TL) indices, hematoxylin-eosin staining, van Gieson staining, qPCR. Results. The alpha-E-catenin dele-
tion leads to a shortened lifespan of the mutant mice. The mice with homozygotic deletion of alpha-E-catenin
had mean survival 36.44 ± 2.62 weeks, haploinsufficiente mice – 38.11 ± 2.39 weeks, controls – 66.2 ±
4.1 weeks. The maximal lifespan in homozygotic mice was 48 weeks, in haploinsufficiente – 44 weeks,
whereas in the control we observed more than 78 weeks. In our experiment we revealed the increasing of HW/
BW and HW/TL indices in the homozygotic and haploinsufficiente mice compared to the control mice. Using
histological staining we found that the full ablation and deficiency of alpha-E-catenin led to the growing of
interstitial fibrosis and cardiomiocyte disintegration. We analyzed the expression of target genes of the ca-
nonical WNT signaling and HIPPO-signaling. Conclusions. We have shown that embryonic cardiospecific
deletion of one as well as both alleles of the alpha-E-catenin gene leads to the disorders of heart structure,
which are typical for the dilated cardiomyopathy, ischemic heart disease, accompanied by fibrosis. As a result
this violation of the heart tissue structure causes an early death of animals. We assume that the loss of alpha-E-
catenin leads to such dramatic consequences not only due to the violation of cells interactions, but to the de-
regulation of signalling pathways in cardiomyocytes, that should be clarified by further molecular genetics and
physiological studies.
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2016. Vol. 32. N 5. P 395–405
mailto:kostikov1212@gmail.com
396
Assessing the relationship between enhancer transcription and activity
during embryonic development
V. Bondarenko, O. Mikhaylichenko, D. Harnett, I. Schor, E. Furlong
European Molecular Biology Laboratory (EMBL), Genome Biology Unit,
Heidelberg, Germany
Aim. Although enhancer transcription has been discovered and extensively studied in mammals, it remains
largely unexplored in Drosophila. As a model organism, Drosophila offers unique advantages over other
organisms for a direct experimental validation of enhancers, including accurate spatio-temporal maps of
enhancer activity during the embryonic development. Given the essential role of enhancers in driving the
transcriptional programs regulating development, the understanding of enhancer transcription and its
contribution to the enhancer activity may offer new insights into the principles of gene regulation, cell identity
control, and development. Methods. Here, we use the Drosophila development as a model system to test the
association of initiating and elongating forms of RNA Polymerase II (Pol II) in various phosphorylation states
with spatial and temporal enhancer activity, over a time course covering the embryonic mesoderm and nervous
system development. We complement our analysis with the strand-specific PRO-cap data, which maps nascent
transcripts to their initiation sites. Resuts and Conclusions. Our results confirm the existence of enhancer
transcription in D. melanogaster at comparable levels to the vertebrate genomes. We also provide evidence for
a bidirectional pattern of the intergenic enhancer transcription similar to the vertebrate enhancers, along with
the presence of initiating Pol II at enhancers. Considering the density of Drosophila genome, we show that
many enhancers are associated with active transcription as a consequence of their genomic location, are the
result of transcriptional read through, or are possible alternative transcription start sites. We also identified an
association between the experimentally defined enhancer activity and enhancer transcription, and showed that
this association was weaker than the vertebrate studies suggest.
397
X Conference of Young Scientists Institute of Molecular Biology and Genetics NAS of Ukraine 26-27 May 2015
mTOR-signaling network in paracryne regulation of HeLa cancer cell
motility by NIH 3T3 fibroblasts
N. Ya. Gotsulyak, A. I. Khoruzhenko
Institute of Molecular Biology and Genetics NAS of Ukraine,
Kyiv, Ukraine
Aim. The main aim of this work was to evaluate the involvement of mTOR signaling in paracrine regulation
of HeLa cancer cell motility by NIH 3T3 fibroblasts. Methods. Cell culture, immunoblotting, immunocyto-
chemistry, soft agar colony formation, 3D-2D cell culture transformation, confocal microscopy, scratch test
assay and Transwell test were used in this study. Results. The significant changes were revealed in malignant
phenotype of HeLa cells cultivated in the NIH 3T3 fibroblasts conditioned medium (CM). The revealed phe-
notypic changes caused by this factor are present at the molecular level as well as at the cellular one. It was
manifested as an increase in the mTORS2448, p85-S6K1T389, p70-S6K1T389, p70-S6K1S371 and FAKY925 phos-
phorylation rate and a decrease in the cell migration activity. The alteration in protein expression profile of the
above mentioned molecules was not found. Besides, the effects of specific mTOR kinase inhibitor and anti-
cancer drug rapamycine on the CM modified HeLa phenotype were investigated. It was shown that the addi-
tion of rapamycin to CM significantly reduced the mTORS2448, p85-S6K1T389 and p70-S6K1T389 phosphoryla-
tion induced by CM. Interestingly, a decline of the mentioned kinases phosphorylation level caused by rapa-
mycin was more pronounced in the cells stimulated with CM as compared to the unstimulated cells. At the
cellular level, a combined treatment with rapamycine and CM enhanced the inhibitory effect on the HeLa cell
migration activity. Moreover, the observed effects of CM, rapamycine and their combination on the cancer cell
motility tightly depended on the cell nutrient consumption. The effect of all these factors as well as the migra-
tion activity decreased under the starvation conditions. Additionally, we also revealed the cytoskeleton remod-
eling, namely, a decrease in cytokeratin branching in HeLa cells, exceptionally under the combined action of
rapamycin and NIH 3T3. The revealed NIH 3T3 CM induced effects on HeLa cells and their sensitivity to the
mTOR inhibition by rapamycine along with the data of other authors give grounds for assuming that mTOR
signaling may play an editing role in the cell reactions and facilitate the inhibition of cancer cell motility by
NIH 3T3 CM. Conclusion. mTOR signaling is involved in the motility regulation of HeLa cells by NIH 3T3
fibroblasts at the molecular, cellular and sub-cellular levels.
398
Prediction of regulatory motifs for RNA-binding proteins in 3’UTRs
of some endocytic genes
A. Hubiernatorova, D. Gerasymchuk
Institute of Molecular Biology and Genetics NAS of Ukraine,
Kyiv, Ukraine
Introduction. The clathrin-mediated endocytosis is fundamental for the neurotransmission, signal transduc-
tion and regulation of many plasma membrane activities. In the first step of endocytosis the interaction be-
tween FCHO1/2, EPS15 and ITSN1/2 is essential. These proteins have different isoforms (between 2 and 4)
encoded by multiple transcripts with variable 3’ untranslated regions (3’UTRs) length (between 262 and 11561
bases). The aim was to predict the sites for regulatory elements in 3’UTRs of FCHO1/2, EPS15 and ITSN1/2,
which may be common for all or the majority of their transcripts and thus may regulate the expression of genes
involved into the first step of endocytosis. Materials and Methods. The analysis was performed by
‘RegRNA 2.0’ and ‘Scan for Motifs’ services. Results. The analysis showed 13 types of regulatory elements.
As far as the FCHO1 transcript variants have very short 3’UTRs (262-270 nt), there are no regulatory elements
predicted for these mRNAs, except 15-LOX-DICE element, which specifically inhibits the 15- Lipoxygenase
gene expression (‘Scan for Motifs’). It is unknown yet whether it regulates the expression of any other genes.
According to ‘Scan for Motifs’, Pumilio binding element (PBE) is common for all FCHO2, EPS15 and ITSN2
transcript variants and for the ITSN1-L transcript variant. Pumilio is a sequence-specific RNA-binding protein
(RBP) that acts as a post-transcriptional repressor by binding the PBE. It mediates the post-transcriptional re-
pression of transcripts by different mechanisms: acts via a direct recruitment of the CCR4-POP2-NOT dead-
enylase leading to the translational inhibition and mRNA degradation, or mediates the deadenylation-inde-
pendent repression by promoting an accessibility of miRNAs. According to both sources, Musashi binding
element (MBE) is common for all FCHO2, EPS15 ITSN1 and ITSN2 transcript variants (is not predicted only
for ITSN1-L isoforms by ‘Scan for Motifs’). The Musashi family is an evolutionarily conserved group of
RBPs, which have emerged as a key signal that confers and protects the stem cell functions during the develop-
ment and regenerative processes. Musashi1 acts as a translational repressor through the sequence-specific in-
teraction with the 3′UTR of various target mRNAs. Conclusion: The obtained data may be used as a basis for
further studies, particularly, for the validation of predicted common sites for RBPs in 3’UTRs of the genes
involved in the first step of clathrin-mediated endocytosis.
399
X Conference of Young Scientists Institute of Molecular Biology and Genetics NAS of Ukraine 26-27 May 2015
In silico study of the complexes ofB.taurus tyrosyl-tRNA synthetase
with substrates
V. O. Kravchuk 1,2, O. V.Savytskyi1, K. O. Odynets1, V. V. Mykuliak1,3, A. I. Kornelyuk1,3
1 Institute of Molecular Biology and Genetics, NAS of Ukraine,
Kyiv, Ukraine;
2 National Aviation University,
Kyiv, Ukraine;
3 Institute of High Technologies, Taras Shevchenko National University of Kyiv,
Kyiv, Ukraine
kravchukvladyslav@gmail.com
Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzyme of protein biosynthesis. At present, the crystallo-
graphic data for the mammalian TyrRS in complexes with the substrates have not been obtained. The struc-
tural exploration of this enzyme is not only of fundamental, but also of biomedical significance. However,
there are few structural data for mammalian TyrRS compared to those] for bacterial enzymes. Aim. In this
work, using several computational modeling techniques, we constructed 3D models of BostaurusTyrRS
(BtTyrRS) structure corresponding to the different stages of the catalytic reaction. To understand the confor-
mational changes involved in the catalytic mechanism, we performed several 100 ns molecular dynamics
(MD) simulations for separate complexes. Methods. The previously reported full-length HsTyrRS structure
(Savytskyi et al., 2013) was used as a template for the full-length BtTyrRS structure modeling. We constructed
the catalytic loop in different conformations with subsequent ligand docking. Modeller 9 and AutoDock 4.2.6
software were used for molecular modeling and docking, respectively. The selected protein-ligand complexes
were simulated for 100 ns using all-atom MD simulations with GROMACS 5.0 (CHARMM27 force field).
Results. It was found that ATP binds at the active site via hydrogen bonds interactions with more than 50 % of
simulation time (50–100 ns): Val215 – 96 %, Asn212 – 86 %, Trp40 – 85 %, Lys154 – 78 %, Lys222 – 62 %,
and Tyr52 – 56 % whereas the same interactions [of ?] fortyrosyl-adenylate (Tyr-AMP) are following:Ala43 –
99 %, Thr42 – 99 %, Asp173 –98 %, Tyr39 – 97 %, Asn212 – 77 %, Val215 – 73 % and Trp40 – 63 %. For the
TyrRS complexes with the Tyr-AMP and the diphosphate ion (PPi), different catalytic loop conformations
were modeled. ThePPi remained in the active site with the compact form of catalytic loop during 100 ns simu-
lation time, whereas it is rapidly (14 ns) released from the active site with the extended form of catalytic loop.
We revealed that both Mg2+and K+ were functionally crucial in each step of the catalytic reaction. Conclusion.
We performed the modeling of the BtTyrRS complexes with the substrates corresponding to different stages of
the catalytic reaction with the following MD simulations. The results obtained are in good agreement with
other experimental and modeling data which allows usage of these models for the study on the mechanism of
mammalian TyrRS function.
Acknowledgments. This work was supported by NAS of Ukraine (grants 15/2010–2016). Authors would like
to thank Tukalo M.A. for discussions and Ukrainian national grid-infrastructure administration for technical
support.
mailto:kravchukvladyslav@gmail.com
400
An iron (II) clathrochelate derivatives and serum albumins: exploration
of the binding
M. Kuperman1, V. Kovalska1, M. Losytskyy1, O. Varzatskii2, S. Yarmoluk1
1 Institute of Molecular Biology and Genetics,
NASU
2 Institute of General and Inorganic Chemistry,
NASU
mvkuperman@gmail.com
The macrocyclic cage metal complexes –iron(II) clathrochelates possess a range of bioactive properties. These
compounds are able to inhibit T-7 RNA polymerase, suppress the amyloid fibril formation, display a high
toxicity in leukemia cells, bind serum albumins [1]. This provokes interest in studying their interaction with
biomolecules, particularly the proteins. Aim. To characterize the interactions between clathrochelates, bearing
different number of functional groups, with serum albumins (bovine/human, BSA/HSA) by physico-chemical
Methods: fluorescent and circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC).
Results. The protein fluorescence quenching studies evidenced to the complex formation between the func-
tionalized clathrochelates and albumins. This binding strongly depends on the nature of the ribbed substituents
in the clathrochelate framework, an extreme binding affinity is observed for the compounds bearing carboxy
groups (quenching of the BSA fluorescence up to 17 times). Upon binding, clathrochelates are able to gain
optical activity and induce the pronounced CD-signal in 350-600 nm region. The shape and intensities of these
CD-bands are determined by the nature and number of clathrochelate’s ribbed substituents and kind of a pro-
tein. Only an alteration of isomery of carboxyphenylsulfid substituent results in the variations of their CD-
bands intensity upon binding to HSA up to 63 times. In the presence of BSA and HSA, hexa carboxyphenyl-
sulfid substituted clathrochelates evoke the CD-spectra of distinct maxima positions and intensity. Fluorescent
studies at different pH and displacement method show the involving of albumins main binding sites I and II in
this interaction. According to the ITC data, the thermodynamic parameters of the complex formation between
hexa carboxyphenylsulfid clathrochelates and BSA are determined by the isomery of ribbed substituent’s of
clathrochelates. The binding constants of such complexes are moderate, Ka ranges from 5 to 28 103M-1 and the
protein-ligand binding ratio is about 1:2 for meta- and ortho- isomers and 1:1 for para- isomer of compound.
Conclusions. We suggest that the structure of albumin-clathrochelate complex noticeably depends on the
spatial geometry of the clathrochelates ribbed substituents. This is reflected in the optical response –the iso-
mers of functionalized clathrochelates differently affect the protein intrinsic fluorescence and the induced
CD-signal character. This high structure-sensitivity gives clathrochelate the ability to distinguish the similar
proteins (BSA and HSA) by acquiring different CD-bands in their presence.
1. Blechinger J, Varzackii O, Kovalskaet V et al., Bioorg Med Chem Lett, V 26, Iss 2, Pp 626–629(2016)
mailto:mvkuperman@gmail.com
401
X Conference of Young Scientists Institute of Molecular Biology and Genetics NAS of Ukraine 26-27 May 2015
Oligoribonucleotide effects on the influenza virus in vitro and expression
of the nos2, arg2, xdh, nfkbia, nfkb1 genes at the influenza virus infection
in vivo
N. S. Melnichuk, A. O. Rybenchuk, G. V. Gerashchenko, V. I. Kashuba, L. I. Semernikova,
T. G. Yakovenko, Z. Yu. Tkachuk
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
Kyiv, Ukraine
natalia.melnichuk8@gmail.com
Influenza (flu) virus causes activation of NOS2, Arg2, XOD enzymes involved in the production of free radi-
cals, such as NO., O2
.-, ONOO-. Free radicals, involved in signal transduction pathways, activate the transcrip-
tion factors such as NFκB and may lead to the lung tissue damage. It was shown that NFκB induces transcrip-
tion of the proinflammatory genes in influenza-infected cells and takes part in the flu virus life cycle. Natural
and synthetic oligoribonucleotides (ORN) have a wide range of biological effects playing a key role in the
antiviral activity. However, the mechanisms underlying ORN effects are not clear for today. Aim. To study
ORN effects on the influenza virus in vitro and the expression of the nos2, arg2,xdh, nfkbia, nfkb1 genes at
influenza virus infection in vivo. Methods. Experimental groups: healthy – healthy mice; prevention with
ORN – ORN are injected to the mice 24 hours before the flu virus infection; treatment with ORN – ORN are
injected to the mice 24 hours after the flu virus infection; inluenza –the flu-infected mice; control ORN – ORN
are injected to the healthy mice. The ORN were modified by D-mannitol. The genes expression in the mouse
lung cells was investigated with RT-PCR. The gapdh was used as a reference gene. The infectivity of flu virus
and hemagglutinin (HA) activity, determined by cytopathic effect and HA assay respectively, were studied
after incubation with the ORN. Results. The overexpression of all investigated genes was shown in the virus-
infected mice compared to the healthy ones. We observed increasing the mRNA expression level of the xdh,
arg2, nos2, nfkbia and nfkb1 genes in the infected animals by 2.5, 3.5, 10, 3.2 and 1.5 times respectively com-
pared to the healthy animals. A decrease in the mRNA level of the arg2, nfkbia and nfkb1 genes was detected
after the prevention and treatment with ORN compared to the virus-infected mice. The ORN injection for
prevention and treatment reduced the mRNA expression level of the arg2 gene by 50 % and 22 % respec-
tively vs. the virus-infected mice, whereas the mRNA expression of the xdh gene remained unchanged. A de-
crease in the mRNA expression of the nos2 gene by 36 % occurred when the ORN were injected for prevention
and an increase by 73 % at the treatment. The study of ORN influence on the flu virus showed a decrease in
the infectious titer of flu virus by 3.0 lgTCID50 and a 4-fold decrease in HA activity compared to thecontrol
virus (without ORN). Conclusions: Our results have shown that the modification of ORN by D-mannitol
modulates the expression of the nos2, arg2,xdh,nfkbia, nfkb1 genes at the flu virus infection in vivo and affects
flu virus in vitro. We supposed that ORN reduced the flu virus infectivity via decreasing HA activity.
mailto:natalia.melnichuk8@gmail.com
402
Study of genomic variation in Deschampsia antarctica Desv. (Poaceae)
from the maritime Antarctic
D. Navrotska, M. Twardovska, I. Andreev, K. Spiridonova, V. Kunakh
Institute of Molecular Biology and Genetics NAS of Ukraine
Kyiv, Ukraine
navrotska.daria@gmail.com
Aim. Deschampsia antarctica Desv. is a unique species in the family Poaceae that occupies the Antarctic
Peninsula region and demonstrates adaptation to the local unfavorable environments. It is well known that the
stressful environmental factors can influence the plant genome by causing changes in the chromosome number
and structure, as well as an increase in the genetic variation. Therefore, our study was aimed at investigating
alterations in the genome of D. antarctica plants at both chromosomal and molecular levels that may be an
important source of adaptive variation within the species. Furthermore, the genome stability was studied in the
micropropagated plants and tissue cultures of the species. Methods. In the study we used in vitro grown
D .antarctica plants that were derived from the seeds collected in the Argentine Islands region (Darboux,
Galindez, Skua, Great Yalour Islands, and Rasmussen Cape) of Maritime Antarctic. The chromosome number
of plants was determined in the cells of root apical meristems counterstained by DAPI. The 5S rDNA and 25S
rDNA loci were visualized by the dual-color fluorescence in situ hybridization (FISH) technique. For cyto-
logical analysis the samples of tissue cultures derived from the root explants were fixed in ethanol : acetic
acid = 3:1 and stained with 1% aceto-orsein. A molecular-genetic analysis was performed by PCR with poly-
morphic ISSR-primers. Results. Most of D. Antarctica plants from the Argentine Islands region have 2n = 26
chromosomes in their karyotypes. Moreover, the mixoploid plants were also found: accessions from Darboux
Island (2n =13-27, 2n=26+1-2B) and Great Yalour Island (2n =13–39). FISH analysis revealed ten 5S rDNA
sites and four 25S rDNA sites in the plant with the typical chromosome number (2n = 26). However, a greater
number of rDNA loci (twelve of 5S rDNA and six of 25S rDNA) were found in the near-triploid plant from
Great Yalour Island. The molecular-genetic analysis of D. antarctica plants demonstrated that the differences
between the normal and mixoploid plants do not exceed the level of within population variation observed in
the Argentine Islands region. The cytogenetic analysis revealed that tissue cultures have modal class composed
of the cells with diploid and near-diploid chromosome number that indicates the conservation of cytogenetic
features of D. antarctica genotypes at the early stages of culture irrespective of the karyotype of initial plant
(diploid, mixoploid or poliploid). We failed to reveal the genetic changes in the clonally propagated D. antarc-
tica plants during prolonged cultivation in vitro at both molecular (using ISSR-PCR) and chromosomal levels.
The obtained results indicate that in vitro conditions used for the plant culture may ensure maintenance of the
genome stability of D. antarcica. Conclusions. The cytogenetic analysis of D. antarctica plants revealed the
intraspecies chromosomal polymorphism, which was further confirmed by FISH analysis. The molecular ge-
netic analysis of the plants with different chromosome number demonstrated a low genetic diversity. The tissue
cultures obtained from D. antarctica plants with the different karyotypes have similar cytogenetic structure
with a predominance of diploid cells in the proliferative pool.
mailto:navrotska.daria@gmail.com
403
X Conference of Young Scientists Institute of Molecular Biology and Genetics NAS of Ukraine 26-27 May 2015
Molecular basis for proposed mechanism of editing activity by D-amino-
acyl-tRNA-deacylase from Thermus thermophilus
M. Yu. Rybak, O. P. Kovalenko, M. A. Tukalo
Department of Protein Synthesis Enzymology, Institute of Molecular Biology and Genetics, NAS of Ukraine
Kyiv, Ukraine
Introduction. Quality control during protein biosynthesis (particularly – in translation) provides an accurate
flow of genetic information from mRNA to the correct amino acid sequence. This process is controlled by
several mechanisms: precise amino acid recognition by aminoacyl-tRNA-synthetases (aaRSs), proofreading
mechanisms against appearing mistakes by cis- (aaRSs) and trans-editing factors (additional enzymes), and
discrimination of mischarged substrates by the elongation factors. D-aminoacyl-tRNA-deacylase (DTD) be-
longs to such trans-factors. It is involved in the correction of mischarged tRNAs with D-amino acids by aaRSs,
which are deficient in the L-stereospecificity in recognition process. DTD is specific only to D-aminoacyl-
tRNA substrates (D-Tyr/D-Phe/D-Trp/D-Asp-tRNA) and does not hydrolyse L-aminoacyl-tRNAs. TyrRS is
not able to discriminate the stereospecificy of amino acids, requiring an additional checkpoint before elonga-
tion. Aim. To investigate the mechanism of aminoacylation by TyrRS from Thermus thermophilus (TyrRSTT)
and editing misaminoacylated D-Tyr-tRNATyrby DTD from T. Thermophilus (DTDTT), we used molecular
modelling, molecular dynamic (MD) simulations, site-directed mutagenesis of the enzyme and tRNATyr mod-
ifications. Methods. We performed comprehensive site-directed mutagenesis studies, based on molecular
modelling and MD simulations of the proposed active site of DTDTT and amino- and deacylation assays with
α-[32P]-tRNATyr from T. thermophilus. The structural model of DTDTT bound to the D-Tyr-A76 was gener-
ated by homology modelling using the reported crystal structure of Plasmodium falciparum DTD bound to
this substrate [Ahmad, eLife, 2013]. MD simulations were used to study the frames where the water molecules
formed a necessary angle and distance to perform a nucleophilic attack at a carbonyl group of the ester of the
mischarged D-Tyr-tRNATyr . The assays with radiolabelled tRNATyr were applied as an experimental basement
of the proposed catalytic mechanism. Results. The kinetic parameters for TyrRSTT aminoacylation reaction
were determined from Michaelis-Menten plot by Origin 9.0 software, representing the average values from at
least three independent measurements. The results of MD simulations after 5 ns were analysed to perform
further in site-directed mutagenesis of the enzyme’s active site. DTDTT and its substitution mutants G137A
and P138A showed significant differences in the activity, confirming the importance of Gly-Pro motif in the
enzyme’s selectivity. The role of 3’-OH group of the terminal ribose of tRNATyr is also essential for DTDTT’
proofreading mechanism of the misaminoacylated substrates. Conclusions. In sum, because of the absence of
discrimination between L- and D-Tyr by TyrRS (discrimination factor is only 11 against 3300 in the literature
data of the ratio cognate/noncognate amino acid), there is the necessity of additional checkpoint of D-aminoacyl-
tRNA substrates by DTDTT, the catalytic mechanism of which, based on MD stimulations and primary mu-
tagenesis studies is proposed here.
404
Molecular docking study of oligoribonucleotides with D-mannitol
molecule
V. Shchodryi, D. Lozhko, Z. Tkachuk
Institute of Molecular Biology and Genetics, National Academy of Sciences
shodryj1992@gmail.com
Aim. RNA is a pharmacologic target of many drugs currently used in clinical trials. Small molecules that bind
RNA have been proven to be effective anticancer, antibiotic, and antiviral therapeutic agents. One of the most
effective drugs of this series is an antiviral and anti-inflammatory drug “Nucleks”. In our spectrometric studies
we have found that RNA, which is part of the drug, forms complex with mannitol. Accordingly, the aim of our
research was to model the complex formation between oligoribonucleotides and D-mannitol molecule. Also,
we present the application of the flexible ligand docking techniques in order to elucidate the most probable
oligoribonucleotide binding mode with D - mannitol molecule. Methods. Our investigation was performed
using “Autodock”, “Hyperchem” and “Chimera” software packages. Results. We have studied the interaction
of oligoribonucleotides with different nitrogenous bases: adenine, uracil, guanine and cytosine. For complex
formation we used oligoribonucleotides with 1, 3, 5 10, 15 nucleotides. We have obtained complex spatial
structure of D-mannitol molecule with oligoribonucleotides. We have found that binding energy of single nu-
cleotides is different and lies within the range of -2.0 to -1.9 kcal / mol. For 3 residue-long oligoribonucleotides
the binding energy lies in the range of -4.1 to -2.7 kcal / mol. In this case the binding energy for oligoadenilate
with D-mannitol is significantly less in comparison to binding energies for other oligoribonucleotides. For
5 residue-long oligoribonucleotides the bindig energy lies in the range of -3.9 to -3.4.For 10 residue-long oli-
gorybonucleotides the binding energy value lies in the range of -4.0 to -3.3 kcal / mol. The lowest binding
energy was observed in case of oligoguanilate. For 15 residue-long oligoribonucleotides the binding energy
value was in the range of -4.3 to 3.1 kcal / mol. Similarly to previous cases, the biggest binding energy was
observed in case of oligoguanilate. Therefore, the molecular docking analysis suggests that the D-mannitol
molecule binds to the nitrogenous base within the oligoribonucleotide. Conclusion. This study allows us to
predict more accurately the nature of the interactions between the molecules D–mannitol and oligoribonucleo-
tides.
mailto:shodryj1992@gmail.com
405
X Conference of Young Scientists Institute of Molecular Biology and Genetics NAS of Ukraine 26-27 May 2015
Conformational changes of Interferon under the influence
of oligoribonucleotides and their derivative
M. M. Vivcharyk1, M. S. Iakhnenko1,2, S. M. Levchenko1,3, S. I. Chernykh1, Z. Yu. Tkachuk1
1 Institute of Molecular Biology and Genetics of NASU,
2 Taras Shevchenko National University
3 College of Optoelectronic Engineering Shenzhen University,
China
vivcharykma11@rambler.ru
Aim. RNA-based antiviral drugs are actively implemented in practical medicine during last decades, neverthe-
less the molecular mechanism of their action is still unclear. As it was shown in our previous work the combi-
nation of total yeast RNA with alcohol sugar D-mannitol leads to the changes of the biological activity and
efficiency. At this stage of our investigation we studied the ability of total yeast RNA and RNA-D-mannitol
complex to affect the conformation and stability of interferon (IFN) α-2b - a key protein of the antiviral cell
defense mechanism. Methods. To investigate the interaction, conformational changes and stability of IFN
protein at the presence and/or absence of the ligands the fluorescence and CD (circular dichroism) spectrosco-
pies were used. All experiments were performed on spectrofluorometer Jasco FP-8200 and CD spectrometer
Jasco J-815 with the peltier temperature cell holder. Results. The thermal denaturation profiles of IFN α–2b,
IFN α–2b (PEG) and IFNα–2b with 100mM NaCl alone and in the presence of RNA and RNA-D-mannitol
complex were obtained from their fluorescence spectra at the temperature range of 25-75 °C. The analysis of
these profiles shows that the addition of ligands leads to the thermal stabilization of protein and could indicate
the interaction between IFN α-2b and the mentioned compounds that causes the changes of its conformation.
For further confirmation of this assumption we calculated a dissociation constant that appeared to be relatively
weak (micromolar) in all cases. CD spectroscopy demonstrated that both RNA and RNA-D-mannitol complex
caused tiny alterations in the IFN 3D structure reflected in a small decrease in molar ellipticity within both
helical bands. The analysis of IFN secondary structure changes by CDNN software shows that adding RNA or
RNA-D-mannitol complex leads to the decreasing of α-spiral components in the protein structure and to the
small increasing of β-turn and random coil components. All studied probes demonstrated that RNA-D-
mannitol complex affects the IFN α-spiral stronger than RNA itself. Conclusions. We suppose that total yeast
RNA and RNA-D-mannitol complex act as compounds, altering the secondary structure of interferon and in
this way can change its biological activity. Our results are important for revealing a molecular mechanism of
the antiviral action of RNA-based compounds.
mailto:vivcharykma11@rambler.ru
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| id | nasplib_isofts_kiev_ua-123456789-152849 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T18:46:44Z |
| publishDate | 2016 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | 2019-06-13T08:24:15Z 2019-06-13T08:24:15Z 2016 Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU // Вiopolymers and Cell. — 2016. — Т. 32, № 5. — С. 395-405. — англ. 0233-7657 https://nasplib.isofts.kiev.ua/handle/123456789/152849 en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Chronicle and Information Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU Матеріали Х щорічної конференції молодих вчених Інституту молекулярної біології і генетики НАНУ Материалы X ежегодной конференции молодых ученых Института молекулярной биологии и генетики Article published earlier |
| spellingShingle | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU Chronicle and Information |
| title | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU |
| title_alt | Матеріали Х щорічної конференції молодих вчених Інституту молекулярної біології і генетики НАНУ Материалы X ежегодной конференции молодых ученых Института молекулярной биологии и генетики |
| title_full | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU |
| title_fullStr | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU |
| title_full_unstemmed | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU |
| title_short | Materials of X annual Conference of Young Scientist of the Institute of Molecular Biology and Genetics NASU |
| title_sort | materials of x annual conference of young scientist of the institute of molecular biology and genetics nasu |
| topic | Chronicle and Information |
| topic_facet | Chronicle and Information |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/152849 |