Basic research
Mutations alternating the dynamics of the N-terminal domain of the RyR2 channel

Authors: Bauer, J., Borko, Ľ., Pavlovič, J., Kutejová, E., Bauerová-Hlinková, V.

Human ryanodine receptor 2 (hRyR2) is a calcium channel essential for the release of Ca2+ from the sarcoplasmic reticulum into the cytoplasm of myocytes, thereby ensuring regular cardiac activity. Several mutations have been found in the RYR2 gene, which are responsible for disrupting the function of this channel, leading to life-threatening diseases - heart arrhythmias. One of the mutational clusters is located in the N-terminal domain (NTD) of hRyR2, residues 1-655. Using molecular dynamics, we characterized three mutations located in the central helix of NTD hRyR2: R414L, I419F and R420W, which are responsible for CPVT1 and ARVD2 cardiac arrhythmias. The results indicate relatively small changes in the tertiary structure of hRyR2 that were caused by the introduction of mutations, but a significant change in the opening/closing dynamics of the hRyR2 channel. A change in the dynamics of hRyR2 very likely affects the regulation of calcium flow in muscle cells, thereby significantly contributing to the occurrence of cardiac arrhythmias.


Projects:
VEGA 2/0140/16 - Štruktúra a funkcia RIH domén ľudského ryanodínového receptora 2, ich interakcii s ligandami ako základ pre vývoj liečiv v terapii srdcových arytmií (Structure and function of RIH domains of human ryanodine receptor 2, their interaction with ligands as a basis for drug development in the treatment of cardiac arrhythmias)
Publications:

1. BAUER, J. - PAVLOVIĆ, J. - BAUEROVÁ-HLINKOVÁ, V. Normal mode analysis as a routine part of a structural investigation. In Molecules, 2019, vol. 24, no. 15, p. 3293. (2018: 3.060 - IF, Q2 - JCR, 0.757 - SJR, Q1 - SJR)
2. BAUER, J. - PAVLOVIĆ, J. - BAUEROVÁ-HLINKOVÁ, V. Normal mode analysis as a routine part of a structural investigation. In Molecules, 2019, vol. 24, no. 15, p. 3293. (2018: 3.060 - IF, Q2 - JCR, 0.757 - SJR, Q1 - SJR)
   

 

Basic research
Characterization of the unique antibiotic auricin in the strain Streptomyces lavendulae subsp. lavendulae

Authors: Kormanec, J., Nováková, R.,Homerová, D., Fecková, Ľ., Řežuchová, B., Csolleiová, D., Bekeová, C., Mingyár, E., Ševčíková, B.

Streptomycetes are dominant producers of secondary metabolites with a wide range of biological activities. We identified the antibiotic auricin in the S. lavendulae CCM 3239 strain. The structural analysis of auricin revealed its unique properties. It is modified with D-forosamine and contains a unique aglycon similar to griseusins, which belong to the pyranonaphthoquines. Auricin is active against gram-positive bacteria and exhibits cytotoxicity against human tumor cell lines. Sequencing of the gene cluster responsible for auricin biosynthesis revealed its unusual organization. It consists of a central region containing biosynthetic genes homologous to angucyclin genes. However, a number of auricin biosynthetic genes are located around this central part and are homologous to pyronaphthoquinone genes. These results suggest a unique and so far unknown mechanism of auricin biosynthesis by two overlapping biosynthetic pathways; for angucyclines and pyronaphthoquinones.


Projects:
APVV-15-0410 - Syntetická biológia pre produkciu nových biologicky aktívnych látok u streptomycét (Synthetic biology for the production of new biologically active substances in Streptomycetes)
Publications:

1. PÄTOPRSTÝ, V. - SASINKOVÁ, V. - UHLIARIKOVÁ, I. - ŠEVČÍKOVÁ, B. - REŽUCHOVÁ, B. - HOMEROVÁ, D. - KORMANEC, J. A structural analysis of the angucycline-like antibiotic auricin from Streptomyces lavendulae subsp. lavendulae CCM 3239 revealed its high similarity to griseusins. In Antibiotics, 2019, vol. 8, no. 3, no. E102. (2018: 2.921 - IF, Q2 - JCR, 1.121 - SJR, Q1 - SJR)
2. KORMANEC, J. - REŽUCHOVÁ, B. - HOMEROVÁ, D. - CSÖLLEIOVÁ, D. - ŠEVČÍKOVÁ, B. - NOVÁKOVÁ, R. - FECKOVÁ, Ľ. Recent achievements in the generation of stable genome alterations/mutations in species of the genus Streptomyces. In Applied Microbiology and Biotechnology, 2019, vol. 103, p. 5463–5482. (2018: 3.670 - IF, Q2 - JCR, 1.127 - SJR, Q1 - SJR)

Basic research
The role of the bee enzyme glucose oxidase (GOX) in the antibacterial activity of honey

Authors: Majtán, J., Farkašovská, J., Godočíková, J., Bugárová, V.

Hydrogen peroxide is the main antibacterial factor in honey, which is generated by the enzymatic conversion of glucose under aerobic conditions in diluted honey. In our study, we focused on the development of a semi-quantification method for determining the amount of GOX enzyme in selected types of honey. The obtained results show that the production of H₂O₂ does not correlate with the amount of GOX enzyme in individual types of honey, and it is likely that other components of honey (polyphenolic substances) are also involved in the generation of H₂O₂. Proteolytic cleavage of the protein content of honey with proteinase K had no negative effect on the overall antibacterial potential of individual types of honey. On the other hand, incubation of honeys with catalase led to a statistically significant decrease in the antibacterial activity of honeys, which demonstrates the key role of H₂O₂ in the antibacterial activity of honey. The fact that the antibacterial activity of honey is preserved even after proteolytic cleavage of the protein content of honey is important for its clinical application in the management of chronic wound healing, which is characterized by a strong proteolytic environment.


Projects:

1. VEGA 2/0004/18 - Vplyv včelieho enzýmu glukózooxidáza na antibakteriálne vlastnosti medu a charakterizácia jeho produkcie a aktivity v podhltanových žľazách včely medonosnej -Apis mellifera (Influence of bee enzyme glucose oxidase on antibacterial properties of the honey and characterization of its production and activity in the pharyngeal glands of honey bee - Apis mellifera)
2. Projekty bilaterálnej spolupráce medzi ÚMB SAV a Slovenským zväzom včelárov:

• Pilotná štúdia monitorovania antibakteriálnej aktivity medov zo Slovenska.
• Stanovenie nových kvalitatívnych parametrov zohľadňujúcich biologické vlastnosti medu.

          (Bilateral cooperation projects between ÚMB SAS and the Slovak Association of Beekeepers:

• Pilot study of monitoring the antibacterial activity of honeys from Slovakia.
• Determination of new quality parameters taking into account the biological properties of honey.)

Publications:
BUČEKOVÁ, M. - JARDEKOVÁ, L. - JURICOVÁ, V. - BUGÁROVÁ, V. - DI MARCO, G. - GISMONDI, A. - LEONARDI, D. - FARKAŠOVSKÁ, J. - GODOČÍKOVÁ, J. - LAHO, M. - KLAUDINY, J. - MAJTÁN, V. - CANINI, A. - MAJTÁN, J. Antibacterial activity of different blossom honeys: New findings. In Molecules, 2019, vol. 24, no. 8, no. 1573. (2018: 3.060 - IF, Q2 - JCR, 0.757 - SJR, Q1 - SJR)
FARKAŠOVSKÁ, J. - BUGÁROVÁ, V. - GODOČÍKOVÁ, J. - MAJTAN, V. - MAJTÁN, J. The role of hydrogen peroxide in the antibacterial activity of different floral honeys. In European Food Research and Technology, 2019, vol. 245, p. 2739–2744. (2018: 2.056 - IF, Q2 - JCR, 0.704 - SJR, Q1 - SJR)

Applied research
Bio-cleaning of animal glue from historical objects using bacterial proteolytic enzymes

Authors: Kisová, Z., Planý, M., Kraková, L., Pangallo, D.

Extracellular enzyme mixtures (EEMs) from three bacterial isolates (Paracoccus sp., Bacillus flexus and Exiguobacterium undae) were used to remove the gum adhesive present on the spine of bound newspapers. FT-IR analysis showed that the glue adhesive was effectively removed by the thiose solution and the EEM mixture. The EEM E. undae mixture achieved the best results. Our study describes a simple strategy for the production and characterization of enzymatic mixtures aimed at the degradation of a specific substrate and their subsequent use for the biocleaning of historical objects.



Projects:
APVV-15-0528 - Modifikované polyméry z obnoviteľných zdrojov a ich degradácia (Modified polymers from renewable sources and their degradation)
Publications:
JESZEOVÁ, L. - BENŽOVÁ, R. - GLUŠTÍKOVÁ, M. - OPÁLKOVÁ ŠIŠKOVÁ, A. - KISOVÁ, Z. - PLANÝ, M. - KRAKOVÁ, L. - BAUEROVÁ-HLINKOVÁ, V. - PANGALLO, D. Biocleaning of historical documents: The use and characterization of bacterial enzymatic resources. In International Biodeterioration & Biodegradation, 2019, vol. 140, p. 106-112. (2018: 3.824 - IF, Q1 - JCR, 1.255 - SJR, Q1 - SJR)

Applied research
Identification of AmyC α-amylase from the GH57 family from Thermotoga martitima as a glycogen branching enzyme with high hydrolytic activity

Authors: Janeček, Š.

AmyC family GH57 glycosidase from Thermotoga maritima MSB8 was originally identified as an intracellular alpha-amylase. Based on a detailed in silico analysis focusing on phylogenetic relationships and tertiary structure, it was suggested that AmyC could essentially be a glycogen branching enzyme. Through biochemical characterization, it was experimentally confirmed that AmyC is indeed a glycogen branching enzyme with relatively high hydrolytic (i.e. alpha-amylase) activity (up to 30% of total activity). It forms branched alpha-glucan with 8.5% alpha-1,6- glycosidic bonds. Other hypothetical glycogen branching enzymes from the Thermotogaceae family, showing high sequence similarity to AmyC, have the same sequence-structural features as AmyC. This suggests, that alpha-glucan branching enzymes from the GH57 family with relatively high hydrolytic activity could be widely distributed in nature.


Projects:
VEGA 2/0146/17 – Evolúcia amylolytických enzýmov (Evolution of amylolytic enzymes)
Publications:
ZHANG, X. - LEEMHUIS, H. - JANEČEK, Š. - MARTINOVIČOVÁ, M. - PIJNING, T. - VAN DER MAAREL, M.J. Identification of thermotoga maritima MSB8 GH57 α-amylase AmyC as a glycogen-branching enzyme with high hydrolytic activity. In Applied Microbiology and Biotechnology, 2019, vol. 103, p. 6141–6151. (2018: 3.670 - IF, Q2 - JCR, 1.127 - SJR, Q1 - SJR)

Applied research
Methods of studying the transcriptional activity of proteolytic genes of lactic acid bacteria during cheese maturation

Authors: Pangallo, D., Kraková, L., Puškárová, A., Bučková, M.

Catabolism of milk proteins in cheese is one of the ways in which microorganisms affect the sensory properties of the final product. In this research, we focused on four genes (prtP, pepX, pepN and bcaT) responsible for the production of volatile aromatic substances from proteins in lactic acid bacteria (LAB). We studied the dynamics of these genes and their respective LAB host during ripening of raw sheep's milk cheese using metagenomics and metatranscriptomics approaches.


Projects:
APVV-14-0025 - Metatranskriptóm ovčieho hrudkového syra: RNA-prístup na určenie príspevku mikroorganizmov k organoleptickej kvalite bryndze (Metatranscriptome of sheep lump cheese: RNA-approach to determine the contribution of microorganisms to the organoleptic quality of bryndza cheese)

Publications:
PANGALLO, D. - KRAKOVÁ, L. - PUŠKÁROVÁ, A. - ŠOLTYS, K. - BUČKOVÁ, M. - KOREŇOVÁ, J. - BUDIŠ, J. - KUCHTA, T. Transcription activity of lactic acid bacterial proteolysis-related genes during cheese maturation. In Food microbiology, 2019, vol. 82, p. 416-425. (2018: 4.089 - IF, Q1 - JCR, 1.402 - SJR, Q1 - SJR)

International research projects
CD222 as a plasminogen receptor in the removal of apoptotic cells

Authors: Petrovčíková, E., Vičíková, K., Leksa, V.

Apoptosis, programmed cell death, is an important mechanism for maintaining homeostasis. Unlike necrosis, apoptosis does not cause inflammation but dead apoptotic cells must be removed from tissues. We have discovered a new mechanism for the removal of apoptotic cells. We have shown that CD222 (receptor for mannose 6 phosphate and insulin-like growth factor 2) is present at a higher level on differentiated human macrophages. These cells are professional phagocytes – they remove not only pathogens during inflammation, but also apoptotic dead cells.


Projects:

1. VEGA 2/0020/17 - Ľudský mliečny bioaktívny glykoproteín laktoferín ako regulátor homeostázy (Human milk bioactive glycoprotein lactoferrin as a regulator of homeostasis)
2. APVV-16-0452 - Regulácia pericelulárnej proteolýzy: od molekulárnych mechanizmov k novým subsetom imunitných buniek a terapeutickým nástrojom (Regulation of pericellular proteolysis: from molecular mechanisms to new subsets of immune cells and therapeutic tools)
3. FWF - Austrian Science Fund (No. P22908) - The Integral Regulatory Role of (CD222) in T cell activation.

Publications:
OHRADANOVA-REPIC, A. - MACHACEK, C. - DONNER, C. - MÜHLGRABNER, V. - PETROVČÍKOVÁ, E. - ZAHRADNÍKOVÁ, A. - VIČÍKOVÁ, K. - HOŘEJŠÍ, V. - STOCKINGER, H. - LEKSA, V. The mannose 6-phosphate/insulin-like growth factor 2 receptor mediates plasminogen-induced efferocytosis. In Journal of Leukocyte Biology, 2019, vol. 105, no. 3, p. 519-530. (2018: 4.012 - IF, Q2 - JCR, 1.929 - SJR, Q1 - SJR)

International research projects
Yeast phosphatidylinositol transfer protein Pdr17 does not require high affinity phosphatidylinositol binding for its cellular function

Authors: Pevala, V., Kotrasová, V., Bauer, J.A., Kutejová, E.

The yeast phosphatidylinositol transfer protein Pdr17 is an essential component of the complex required for the decarboxylation of phosphatidylserine (PS) to phosphatidylethanolamine outside the mitochondria. We generated a mutant Pdr17 protein and showed that it is unable to bind phosphatidylinositol (PI) from membranes of permeabilized cells but is still able to transport PI between two membranes in vitro. Molecular models suggest that the mutations only altered the lipid binding cavity of Pdr17p and not its surface properties. Unlike Pdr16p, a close homologue, the ability of Pdr17p to bind PI is not required for its main cellular function in transmembrane transfer of PS. The ability to bind and transport PI is required for the proper function of Pdr16p, whereas Pdr17p has adapted to a different role that does not require high-affinity PI binding, although the protein retains the ability to transport PI.


Projects:
APVV-0654-15 - Úloha medziorganelových interakcií v lipidovej homeostáze (The role of inter-organ interactions in lipid homeostasis)
Publications:
PEVALOVÁ, Z.- PEVALA, V.- BLUNSOM, N. J. - TAHOTNÁ, D. - KOTRASOVÁ, V. - HOLIČ, R. - POKORNÁ, L. - BAUER, J. - KUTEJOVÁ, E. - COCKCROFT, S. - GRIAČ, P. Yeast phosphatidylinositol transfer protein Pdr17 does not require high affinity phosphatidylinositol binding for its cellular function. In Biochimica et Biophysica Acta : Molecular and Cell Biology of Lipids, 2019, vol. 1864, iss. 10, p. 1412-1421. (2018: 4.402 - IF, Q1 - JCR, 2.342 - SJR, Q1 - SJR)

International research projects
Isolation and characterization of sporulating Gram-positive bacteria that are able to bind and reduce chromium (IV) in heavily contaminated soils

Authors: Barák, I., Chromíková, Z., Muchová, K., Chovanová, R.

Together with partners from Switzerland and Serbia, we continued with isolation and characterization of strains of Gram-positive bacteria that would be suitable for use in the remediation of chromium-polluted areas. As part of this collaboration, we isolated 15 environmental, sporulating Gram-positive bacteria. These were isolated from the soil of various chromium polluted areas. We identified strains from the Bacillus genus, most of them belonging to the Bacillus cereus group. Of these, one was identified to be Bacillus pseudomycoides, which was used for further studies. We continued with genetic studies of selected strains to elucidate the contribution of chromium efflux to the overall chromium tolerance of these strains. We used different protocols to introduce DNA into bacilli belonging to the B. cereus group in order to disrupt the chrA gene on the chromosome of the original organism. At the same time, we cloned the chrA gene with its promoter and regulatory regions from the original strain identified as B. pseudomycoides into Escherichia coli and Bacillus subtilis in order to improve their chromium tolerance. In addition, we also cloned the chrA gene with the promoter and regulatory regions from the collection strain of B. pseudomycoides.
Projects:
Scopes – SNF (Swiss National Foundation) - IZ73Z0_152527 / 1 - Úloha homeostázie kovov, ich redukcia a sporulácia v rezistencii Gram-pozitívnych baktériách (The role of metal homeostasis, their reduction and sporulation in the resistance of Gram-positive bacteria)
Publications:
TAMINDŽIJA, D. - CHROMÍKOVÁ, Z. - SPAIC, A. - BARÁK, I. - BERNIER?LATMANI, R. - RADNOVIĆ, D. Chromate tolerance and removal of bacterial strains isolated from uncontaminated and chromium-polluted environments. In World Journal of Microbiology & Biotechnology, 2019, vol. 35, no. 4, p. 35-56. (2018: 2.652 - IF, Q2 - JCR, 0.743 - SJR, Q2 - SJR)