BOOK OF ABSTRACTS
Recent topics in toxinology
7. 6. 2019 Ljubljana
τοξικόν
Recent topics in toxinology
τοξικόν
[gr. toxicon] "poison for use on arrows"
7. 6. 2019
Ljubljana
RECENT TOPICS IN TOXINOLOGY 2019
Organised by
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry Chair of Biochemistry, Department of Biology, Biotechnical Faculty, University of
Ljubljana
Programme Committee
Gregor
Organising committee
Editors Gregor Anderluh, Katja Pirc,
Technical editors
Gregor Anderluh, Matic Kisovec, Katja Pirc Credits for presentation of toxic organisms to
Gregor Anderluh, , Mojca Mally, Katja Pirc, , Tom Turk for text and , Denis Kunkel, Pixabay, Tom Turk, Bruce Watt (University of Maine) for
images Issued by
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry
@NanoSciNIC @kemijski http://bit.do/toxin2019 Printed by
Infokart d.o.o., Ljubljana Circulation
75 issues
Complimentary publication
CIP -
615.9(082)
RECENT Topics in Toxinology (simpozij) (2019 ; Ljubljana)
Recent topics in toxinology : 7. 6. 2019 Ljubljana / [Recent topics in toxinology 2019 ; organized by Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry [and] Chair of Biochemistry, Department of Biology, Biotechnical Faculty, University of Ljubljana ; editors Gregor Anderluh, Katja Pirc - Ljubljana : Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, 2019 ISBN 978-961-6104-44-9
1. Gl. stv. nasl. 2. Anderluh, Gregor, 1969- COBISS.SI-ID 300251904
3
Welcome
Venoms are a fascinating and rich source of molecules with interesting properties and mechanisms of action. The aim of this symposium is to bring an overview of current efforts on various toxinological topics, spanning from the evolution and structure of toxin molecules, through their physiological effects, to their use in medical and biotechnological applications. Renowned scientists working in the field of natural toxins from Slovenia and abroad will present these exciting topics. This University of Ljubljana, who is one of the doyens of toxinology in Slovenia, and whose research interest was oriented mainly towards membrane-active proteins from Cnidaria.
Historically, modern toxinology in Slovenia was initiated in fifties by a biochemist Prof. Drago Lebez from Ljubljana in collaboration with a Croatian physician, Dr.
nt publication was a monography, his younger collaborators started biochemical characterization of some snake venoms, in particular Vipera ammodytes, and cnidarian neurotoxins and cytolytic toxins. So, it is allowed to say that research of venoms and toxins here in Ljubljana is rather traditional and continuous for more than sixty years.
We are particularly happy that many young researchers will attend the symposium.
We hope that a full day of interesting presentation and stimulating discussions will raise an interest in various toxinological topics in young generation of scientists to continue with excellent stories of toxic molecules that will lead to basic knowledge, as well as useful applications in medicine and biotechnology.
Prof. dr. Gregor Anderluh, P
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Contents
Welcome 3
Sponsors 7
Programme 9
Abstracts - Invited Lectures 13
Abstracts - Oral Presentations 29
Abstracts - Posters 39
List of Participants 55
Author Index 61
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Sponsors
Thank you for your support!
9
Programme
9:00-9:15 Opening of the symposium
Physiological aspects of toxins
/ chair: Cesare Montecucco9:15-9:45 Invited 1 R. Manjunatha Kini
Natriuretic peptide analogues with vasodilatory or renal activities:
Personalized care of heart failure patients 9:45-10:05 Invited 2
A unique anticoagulant snake venom protein offers new perspective in venous thromboembolism therapy
10:05-10:25 Invited 3
Different toxicity of hydrophilic and hydrophobic microcystins on rat astrocytes
10:25-10:45 Invited 4 Tom Turk
APS8, a polymeric alkylpyridinium compound blocks α7 nAChR, induces apoptosis in A549 cells, and delays or prevents growth of human lung adenocarcinoma tumours in SCID mice
10:45-10:55 Oral 1 Katja Pirc
The toxicity of NLPs towards plant plasma membrane 10:55-11:05 Oral 2
Orthocaspases: prokaryotic caspase homologues are regulating the toxin-antitoxin systems in cyanobacteria
Coffe break
Methodology in toxinology
11:30-11:50 Invited 5
A transcriptomic and proteomic study of the nose-horned viper venom 11:50-12:10 Invited 6 Toshihide Kobayashi
Recognition of ceramide phosphoethanolamine by aegerolysins 12:10-12:30 Invited 7
Archaeosmes and cholesterol enriched archaeosomes as a molecular system for studying interactions of cholesterol-dependent cytolysins with membranes
12:30-12:40 Oral 3
Triglyceride-based nanodroplets and their interactions with lipid-
12:40-12:50 Oral 4 Mojca Mally
Elucidating the mechanisms of lipid membrane interactions with various active substances
Lunch
Evolution and application of toxins
14:00-14:20 Invited 8 Michael Richardson
Evolution and development of the venom delivery system of snakes 14:20-14:40 Invited 9
Strong and widespread action of site-specific positive selection in the snake venom Kunitz/BPTI protein family
14:40-15:00 Invited 10 Matej Butala
A cytolethal distending toxin variant from Aggregatibacter actinomycetemcomitans
15:00-15:20 Invited 11
Possible applications of aegerolysin proteins in biomedicine and biotechnology
15:20-15:30 Oral 5 Maria Vittoria Modica
The venom of the white gorgonian Eunicella singularis: novel insights into the evolution of Anthozoan toxins
15:30-15:40 Oral 6
Pore-forming toxin as an operator of logic gates Coffe break
Structure with an emphasis on pore-forming toxins
chair: Gregor Anderluh
16:05-16:25 Invited 12 Mauro Dalla Serra Pore-forming toxins 16:25-16:45 Invited 13 Marjetka Podobnik
A mushroom made by an earthworm: the unique transmebrane β- barrel of lysenin
16:45-17:05 Invited 14 Ioan Iacovache
Aerolysin conformational changes towards pore formation 17:05-17:15 Oral 7 Magdalena Kulma
Structural characterization of the lysenin pre-pore state by hydrogen/deuterium exchange mass spectrometry
17:15-17:25 Oral 8 Mirijam Kozorog
Listeriolysin O tryptophans are involved in cholesterol-rich membrane binding and toxin's hemolytic activity
17:25-17:55 Invited 15 Cesare Montecucco Botulinum neurotoxins 17:55-18:00 Closure
11 List of posters
Poster 1 Sabina Berne APS8 induces apoptosis of the NSCLC model cell line by interfering with the lung cancer signalling pathways
Poster 2 Apolonija Bedina Zavec Vesiculation of mammalian cells in response to listeriolysin O and its mutants
Poster 3 Mojca Dobaja Borak Isolation and characterization of C-type lectin-like proteins (snaclecs) from Vipera ammodytes ammodytes (Vaa) venom inducing transient and reversible thrombocytopenia of functional platelets Poster 4 Interaction between ammodytoxin, a β-neurotoxin
from the nose-horned viper venom, and neuronal mitochondria
Poster 5 Biological role of Pseudomonas aeruginosa RahU protein interaction with lipids
Poster 6 Lipid-binding aegerolysins from entomopathogenic fungi
Poster 7 Anastasija Panevska Aegerolysin-based cytolytic complexes acting through ceramide phosphoethanolamine as potential biopesticides
Poster 8 Molecular mechanisms of action and interplay between three key toxins of Listeria monocytogenes Poster 9 Listeriolysin O mutant (LLO Y406A) eliminates
cancer urothelial cells
Poster 10 L-amino acid oxidases are abundant in higher fungi Poster 11 Tina Snoj Solving the mistery: do NLP proteins really form
pores in the lipid membrane?
Poster 12 Spider venom GsMTx4 and mechanosensitive
channel Piezo1
Poster 13 Cholesterol recognition by a protein domain as observed using in vitro evolution
Poster 14 Oligomerization and visualization of Cyt2Aa toxin from Bacillus thuringiensis on model membrane systems
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Abstracts - Invited Lectures
Natriuretic peptide analogues with vasodilatory or renal activities: Personalized care of heart failure patients
R. Manjunatha Kini
Department of Biological Sciences, Faculty of Science, National University of Singapore (Singapore) Natriuretic peptides (NPs) are potent vasoactive hormones, which maintain pressure volume homoeostasis. All three isoforms of mammalian NPs, namely ANP, BNP and CNP, have a conserved 17-residue ring but with highly variable C-terminal extensions. These peptides confer their functions through binding to three membrane-bound NP receptors (NPRs). ANP and BNP bind to NPR-A, whereas CNP binds to NPR-B, both guanylyl cyclase (GC) linked receptors. NPR-A and NPR-B undergo conformational change upon respective NP binding and lead to the production of intracellular cGMP. Snake venom NPs, although having the conserved NP-ring, exhibit distinct biological activity compared with mammalian NPs due to subtle changes in their sequences. We recently identified a new NP from krait venom (KNP), with an unusual 38-residue long C-terminal tail, which has a propensity to form an α-helix. Deletion mutant studies have revealed the presence of two pharmacophores in KNP, namely Ring and Helix. These functional segments induced vasodilation through orthogonal pathways. Ring, like a classical NP, elevates intracellular cGMP levels through activation of NPR-A with a 10-fold lower potency compared to ANP, while Helix uses NO-dependent mechanisms. By systematic structure-function studies, we were able to delineate the amino acid residues that determine vasodilatory and diuretic functions. Using this new knowledge, we developed two classes of human natriuretic peptide analogues (NPAs): one group of NPAs with only vasodilatory effects without diuretic function and the second group with only diuretic effects without vasodilatory function. The diuretic and hemodynamic effects of these human ANP analogues have been evaluated in anesthetized rat models as well as conscious normal and heart failure sheep models. Such distinct classes of NPAs will be useful in the treatment of distinct classes of ADHF (acute decompensated heart failure) patients.
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A unique anticoagulant snake venom protein offers new perspective in venous thromboembolism therapy
(Slovenia)
Venous thromboembolism (VTE) is the pathological process behind two very serious cardiovascular diseases (CVD), deep vein thrombosis and pulmonary embolism. VTE is the third leading cause of CVD-related mortality, also because no adequate therapy is available.
Consequently, there is an enormous need for new anticoagulants to cure VTE, which would not impose high risk of bleeding on patients. Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as the most suitable therapeutic targets to treat VTE. Recently, we described a glycoprotein from the nose-horned viper (Vipera a. ammodytes; Vaa) venom, VaaSPH-1, structurally a serine protease but without an enzymatic activity, which expresses a potent anticoagulant action in human blood1. We demonstrated that its major target in the blood coagulation system is FVIIIa.
VaaSPH-1 antagonizes the binding of FIXa to FVIIIa so preventing the formation of the intrinsic tenase complex. Anticoagulants with such characteristics are intensively sought as they would be much safer for medical application than contemporary drugs, which frequently induce excessive bleeding and other complications. In this lecture, our journey towards original VaaSPH-1-based new generation of anticoagulants to attenuate thrombus formation and propagation without increasing the risk of bleeding will be described.
1 et al., 2018, Thromb Haemost 118, 1713 1728.
Different toxicity of hydrophilic and hydrophobic microcystins on rat astrocytes
, Klara Bulc Rozman, Damijana Mojca
Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana (Slovenia) Microcystins (MCs) are toxic cyanobacterial peptides. Their potent hepatotoxic action mainly causes acute lethality. Chronic intoxication may result in hepatotoxic, nephrotoxic, cardiotoxic, and neurotoxic effects in mammals, including humans. MCs enter cells via multi- specific organic anion-transporting polypeptides (Oatp). Several Oatps have been located in the mammalian blood-brain-barrier (BBB), suggesting that glial and neuronal cells can be exposed to MCs. It is reasonable to expect that hydrophobic MCs could pass cellular membranes more easily then hydrophilic MCs that can cross cellular membranes only by use of Oatps. Astrocytes are crucially involved in the homeostasis of the central nervous system, and our goal was to assess microcystin toxicity on astrocytes in primary cell culture. MCLR was used as a standard hydrophilic MC. MCLW and MCLF were chosen as representatives of hydrophobic microcystins.
Astrocytes were exposed to 0.5, 2 or 10 µM MCs for 24 h. MTT assay showed that MCLF and MCLW, but not of MCLR, caused significant reduction in cell number and viability.
Cytoskeleton disruption presented as degradation of the glial fibrillary acid protein (GFAP), actin and the tubulin network. Percentage of apoptotic cells assessed by Annexin-V labeling increased, The cultured astrocytes expressed several Oatps facilitating the entry of MCs into astrocytes. Intracellular localization of MCLF and MCLW was proven by anti-Adda primary antibody.
At concentrations used, only hydrophobic MCLF and MCLW, but not MC-LR, caused toxic effects in cultured rat astrocytes. Astrocyte degeneration leading to loss of their function could result from chronic exposure to hydrophobic MCs.
Changes in the astrocyte tubulin network following exposure to MC-LW and MC-LF. /
Bulc Rozman et al., 2017, Toxicol Lett 265, 1-8.
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APS8, a polymeric alkylpyridinium compound blocks α7 nAChR, induces apoptosis in A549 cells, and delays or prevents growth of human lung adenocarcinoma tumours in
SCID mice Tom Turk
Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia)
Alkylpyridinium compound APS8, obtained by synthesis based on the natural poly-APS from marine sponge Rhizoniera sarai, is as a strong antagonist of α7 nAChR and a possible lead for novel therapeutic agents for certain types of lung cancer. We evaluated the mechanisms of APS8 activity in vitro and its toxicity and antitumor effects in vivo. We examined the in- vitro activity of APS8 -apoptosis, cytotoxicity, and viability of A549 lung adenocarcinoma cells. We measured Ca2+ influx into cells and evaluated the effects of APS8 on Ca2+ uptake before and after siRNA silencing of the CHRNA7 gene. Additionally, we determined systemic toxicity and organ-specific toxicity of APS8 in male BALB/c mice, as well as evaluated APS8 antitumor activity against subcutaneous human A549 lung and HT29 colon adenocarcinoma xenografts in female SCID mice. APS8 impaired the viability of A549 cells in a dose- dependent manner and induced apoptosis at micro molar concentrations. Nano molar APS8 caused minor cytotoxic effects, while cell lysis occurred at APS8 >3 µM. Furthermore, Ca2+
uptake was significantly reduced in APS8-treated A549 cells. APS8 was not toxic to mice up to 5 mg/kg i.v., and no significant histological changes were observed in mice that survived APS8 treatment. Repetitive intratumoral injections of APS8 (4 mg/kg) significantly delayed growth of A549 cell tumours, and generally prevented regrowth of tumours, but were less effective in reducing growth of HT29 cell tumours. Observed differences in response to APS8 can be attributed to the number of α7 nAChRs expressed in these cells.
A transcriptomic and proteomic study of the nose-horned viper venom
, Adrijana Leonardi, Tamara Sajevic,
nstitute (Slovenia)
The nose-horned viper (Vipera ammodytes) is the most venomous, and thus medically most important, snake species in Europe. At least four subspecies - ammodytes (Vaa), meridionalis, montandoni and transcaucasiana - have been recognized and found mainly in southern Europe and partly in western Asia, spreading from the northwest to the southeast. The main aim of our study was to identify a complete arsenal of Vaa venom proteins and peptides, to guide the production of a more specific and effective antivenom, and promote structure-based drug design for the treatment of certain cardiovascular, neurological and cancer disorders.
We have shown that a major part (more than 88%, see below) of the Vaa venom transcriptome encodes tripeptide metalloproteinase inhibitors, bradykinin-potentiating peptides and natriuretic peptides (MPi, all three on the same precursor), snake C-type lectin- like proteins (snaclec), serine proteases (SP), P-II and P-III metalloproteinases (MP), secreted phospholipases A2 (sPLA2) and disintegrins (Dis). At the protein level, 57 venom proteins belonging to 16 different protein families have been identified. Four of them - SP, sPLA2, snaclec and MP - account for approximately 80% of all venom proteins and are presumably responsible for the main toxic effects, including haemorrhage, coagulopathy, inhibition of platelet aggregation and neurological disturbance. Our combined analysis approach also revealed an original snake venom protein, similar to other P-III MP, but lacking the entire catalytic MP domain, and thus only consisting of a truncated disintegrin-like (D') and complete cysteine-rich (C) domains. Such venom proteins presumably bind platelets and interfere with haemostasis of the prey.
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Recognition of ceramide phosphoethanolamine by aegerolysins
Toshihide Kobayashi
Laboratory of Bioimaging and Pathologies, CNRS University of Strasbourg (France) Ceramide phosphoethanolamine (CPE), a sphingomyelin (SM) analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells.
We showed that mushroom-derived proteins of the aegerolysin family pleurotolysin A2 (PlyA2), ostreolysinA (OlyA), and erylysin A (EryA) strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to SM/Chol, which was previously reported as a target of the aegerolysins, precluded establishment of the binding kinetics. We showed the enrichment of CPE in the central nervous system of Drosophila larvae using PlyA2-EGFP while CPE in the bloodstream form of the parasite Trypanosoma brucei was detected by EryA-EGFP. These results demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.
Aegerolysin proteins are unique A-B type pore-forming toxins where A subunits bind membrane, followed by the binding of B subunit to A subunit and the formation of the pore.
In addition to the formation of pores, OlyA alone has been reported to exhibit biological activities. Atomic force microscopy (AFM) imaging of OlyA binding on the CPE/Chol bilayer at subnanometer resolution revealed a well-ordered structure of OlyA on the membrane as a result of its crystallization (collaboration with Drs Peter ).
Interestingly, the addition of B subunit (PlyB) to OlyA crystal inhibited the formation of pores. The role of OlyA in the regulation of OlyA-PlyB pores will be discussed.
Bhat et al., 2013, J Lipid Res 54, 2933-2943. / Bhat et al., 2015, FASEB J 29, 3920-3934. / Shirota et al., 2016, Biophys J 111, 999-1007. / Inaba et al., 2019, Sci Rep in press.
Binding of PlyA2-EGFP to helical liposomes made of CPE/PC.
Archaeosmes and cholesterol enriched archaeosomes as a molecular system for studying interactions of cholesterol-
dependent cytolysins with membranes
Na
1, Aden
2, Mirijam Kozorog
2,
2, Nada
3
, Marjetka Podobnik
2, Gregor Anderluh
21Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana (Slovenia) / 2Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia)
/ 3Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia)
Archaeosomes are novel generation of liposomes, which are composed of archaeal lipids.
Archaeal lipids have many unique properties. In eukaryotes and bacteria, glycerol has sn- glycerol-3-phosphate stereoisometry and it is ester linked to acyl chains, whereas archaeal lipids use the opposite glycerol stereoisometry (sn-glycerol-1-phosphate) with ether linked isoprenoid side chains. Archaeal lipids isolated from hyperthermophilic archaeon Aeropyrum pernix K1 were used in our study. The isolated lipids contain in majority two polar lipids with direct linkage of inositol and sugar moieties: approximately 91 mol % of 2,3-di-O- sesterterpanyl-sn-glycerol-1-phospho- - -O-α-D-glucosyl)-myo-inositol (C25,25-archaetidyl [glucosyl]inositol; AGI) and approximately 9 mol % of 2,3-di-O-sesterterpanyl-sn-glycerol-1- phospho-myo-inositol (C25,25-archaetidylinositol; AI)1. Large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and nanodiscs from archaeal lipids with incorporated cholesterol can be prepared. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. The members of cholesterol-dependent cytolysins, listeriolysin O (LLO) and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, the specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol was observed. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology2.
1Ulrih et al., 2007, J Membr Biol 219, 1 8. / 2Rezelj et al., 2018, J Membr Biol 251, 491-505.
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Evolution and development of the venom delivery system of snakes
Michael Richardson
Institute of Biology, Leiden University (Netherlands)
Snakes have evolved a sophisticated venom delivery system consisting of a venom gland and hollow fangs. The fangs are self-replacing. Snakes have also evolved venom: a highly complex mixture of toxins. Finally, two lineages have evolved heat-sensitive pits on their heads. This evolution of this amazing armory raises many questions that my lab has been researching over the last decade using an integrated approach: from genes to phenotype via embryology.
In this lecture I trace some of the selective pressures that have driven the evolution of the snake prey-capture armory, and show how these pressures have acted in some cases by modifying the course of embryonic development itself.
Strong and widespread action of site-specific positive selection in the snake venom Kunitz/BPTI protein family
1
,
21Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana (Slovenia) / 2Department of Molecular and Biomedical Sciences, Stefan
Institute (Slovenia)
S1 family of serine peptidases is the largest family of peptidases. They are specifically inhibited by the Kunitz/BPTI inhibitors. Kunitz domain is characterized by the compact 3D structure with the most important inhibitory loops for the inhibition of S1 peptidases. We analysed the action of site-specific positive selection and its impact on the structurally and functionally important parts of the snake venom Kunitz/BPTI family of proteins. By using numerous models we demonstrated the presence of large numbers of site-specific positively selected sites that can reach between 30 50% of the Kunitz domain. The mapping of the positively selected sites on the 3D model of Kunitz/BPTI inhibitors has shown that these sites are located in the inhibitory loops 1 and
replacements have been found exclusively on the surface, and the vast majority of replacements are causing the change of the charge. The consequence of these replacements is the change in the electrostatic potential on the surface of the Kunitz/BPTI proteins that may play an important role in the precise targeting of these inhibitors into the active site of S1 family of serine peptidases.
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A cytolethal distending toxin variant from Aggregatibacter actinomycetemcomitans
Davor
1, Rok
2, Simon Caserman
3, Adrijana Leonardi
4, Maja Jamnik
3, Zdravko Podlesek
1, Katja Seme
5, Gregor Anderluh
3, Igor
4,6
, Peter
1, Matej Butala
11Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia) / 2Department of Oral Medicine and Periodontology, Faculty of Medicine, University of Ljubljana (Slovenia) /
3Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) /
4 (Slovenia) / 5Institute of
Microbiology and Immunology, Faculty of Medicine, University of Ljubljana (Slovenia) / 6Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of
Ljubljana (Slovenia)
The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B- components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the -188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate -188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained.
Possible applications of aegerolysin proteins in biomedicine and biotechnology
Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia)
Aegerolysin protein family (Pfam 06355, InterPro IPR009413) comprises low molecular (15- 20 kDa), acidic, beta-structured proteins, found in several eukaryotic and bacterial taxa.
Although they appear to be among major proteins secreted by the organisms that produce them, their functions and biological roles remain poorly understood.
The common feature of the aegerolysins is their ability to bind different lipids and lipid membranes. Some aegerolysins can target sphingomyelin/ cholesterol membrane nanodomains, while aegerolysins from the fungal genus Pleurotus preferentially bind to ceramide phosphoethanolamine (CPE), which is the major membrane sphingolipid in invertebrates (particularly insects). Moreover, the genomes of some aegerolysin-producing fungi have nucleotide sequences that encode proteins with membrane-attack complex/
perforin (MACPF) domain. In the presence of a protein with a MACPF domain, fungal aegerolysins can function as bi-component lytic complexes for target cell membranes.
Selected fluorescent fusion derivatives of fungal aegerolysins could be used as useful tools to track raft-like membrane nanodomains composed of sphingomyelin and cholesterol. Moreover, the selectivity of some aegerolysin-based cytolytic complexes for increased membrane sphingomyelin/ cholesterol contents can be exploited for selective killing of urothelial carcinoma cells. Finally, due to their specific interaction with CPE, some cytolytic complexes based on Pleurotus-derived aegerolysins could represent a novel promising class of biopesticides for controlling plant pests.
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Pore-forming toxins Mauro Dalla Serra
Institute of Biophysics, Italian National Research Council (Italy)
Pore-forming toxins (PFT) constitute the largest and best characterized class of protein toxins, accounting for more than 30% of all known bacterial protein toxins. They are potent virulence factors evolved during ancient competition among organisms for defense and/or attack purposes. They have an extremely broad taxonomic distribution being produced by bacteria, fungi, animals and plants. Interestingly, similar structures and modes of action are also adopted by components of the immune system, like perforin and complement, and by antimicrobial peptides.
PFT are able to produce lesions in membranes by drilling poorly selective nanometer-sized holes into the target cell membranes, causing cell death through osmotic imbalance.
According to the secondary structure of their membrane-spanning region, PFT can be categorized into two large classes, alpha- and beta- PFT.
Regarding the pore architecture, two main conformations have been described: a purely proteinic channel or a protein-lipid mixed arrangement. This second case is characterized by the co-presence of lipid and protein elements in the pore walls, therefore, the membrane lamellar structure is destroyed and lipids should bend, assuming a toroidal shape.
Here I will shortly discuss examples of both types of functional pores formed by bacterial and animal toxins, investigated by some biophysical techniques, like electrophysiology, fluorescence, AFM. Experimental evidences supporting the ability to punch proteolipidic nanopores into lipid membranes will be presented.
A mushroom made by an earthworm: The unique transmembrane β-barrel of lysenin
Marjetka Podobnik
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) Pore-forming proteins (PFPs) are produced by organisms from all domains of life and have important physiological roles in attack, defense or developmental mechanisms. They are classified as either - or -PFPs, based on the secondary structure of the pore they form in the target lipid membrane. The two main classes of PFPs are composed of several families, and one of them is the Aerolysin family, whose members disrupt the membranes via formation of -barrel pores. The family expands from bacteria to vertebrates, with many bacterial representatives serving as crucial virulence factors. Lysenin is one of the few described eukaryotic members of this family from the earthworm Eisenia fetida. It is present in the coelomic fluid of earthworms to act defensively against parasitic microorganisms. Lysenin is famous of its a high affinity for sphingomyelin in membranes, and can be used as a tool for visualizing distribution and dynamics of sphingomyelin in cells. Moreover, the pores made by Lysenin have interesting biochemical and biophysical features that originate from its unique three-dimensional structure. In my talk I will present and discuss our structural and functional studies of the Lysenin pore, which provide important insights into the mechanism of pore assembly as well as general features of this family of biological nanopores. These results are not only relevant for understanding of pore formation mechanism by Aerolysin- like PFPs, but also for potential applications of such type of pores in medicine and nanobiotechnology.
27
Aerolysin Conformational changes towards pore formation
Ioan Iacovache
Institute of Anatomy, University of Bern (Switzerland)
Aerolysin, a protein produced by Aeromonas spp, is the founding member of a major class of pore forming proteins. One of the first pore forming toxins to be crystalized in its soluble form, the study of aerolysin has revealed many details into the mode of action of pore forming toxins over the years. Recently, through breakthroughs in cryo electron microscopy, the structure of the oligomeric forms of aerolysin and related aerolysin family members have added to our understanding of this intriguing class of proteins. By studying wild type aerolysin and different mutants we could show details into some of the steps required by a pore forming protein to transform from a soluble protein into a membrane inserted oligomer.
A mutant of aerolysin blocked in the pre-pore state, after the oligomerization but before membrane insertion shows the initial rearrangements. The highlight of this initial interaction is a novel protein fold consisting of two concentric β-barrels tightly kept together by hydrophobic interactions thus explaining the stability of the aerolysin oligomer to denaturing agents. Following oligomerization and pre-pore formation another series of conformational changes lead to a collapse of the pre-pore structure culminating with the folding of an extended transmembrane β-barrel.
Botulinum neurotoxins Cesare Montecucco
Department of Biomedical Sciences and Neuroscience, University of Padova (Italy)
Botulism is a paralysis of peripheral cholinergic nerve terminals caused by botulinum neurotoxins (BoNTs), which are produced by anaerobic bacteria of the genus Clostridium.
These are the most poisonous substances known to humans and are included in the list A of potential bioterrorist weapons. At the same time thanks to scientific and clinical research, they are therapeutics used in millions and millions of doses in humans. Traditionally seven serotypes were known, but recent genomics has already characterized > 40 different BoNTs posing the problem of the reasons behind the evolution of so many botulinum neurotoxins.
Great advances on the mechanism od nerve terminal entry have been recently made. This involves the translocation of a 50 kDa metalloprotease domain from the lumen of an endocytic synaptic vesicle across the membrane into the cytosol, where it cleaves any of the three SNARE proteins. The SNAREs are the core of the nanomachine which mediates the Ca2+- controlled release of neurotransmitters at the synapse. We recently found that a key step for the display of the toxin metalloprotease activity is the reduction of a single disulphide bond operated by a a thioredoxin reductase-thioredoxin system associated to synaptic vesicles.
Consequently, we identified a group of molecules that inhibit this redox system and prevent reduction and paralysis in vivo. We are currently attempting to extend experimentations in human volunteers. More recently we have demonstrated a VAMP specific metalloprotease activity in a BoNT-like toxin discovewered by bioinformatics in the genome of the non pathogenic bacterium Weisella oryzae.
The reduction of the single interchain bond of botulinum and tetanus neurotoxins.
Abstracts - Oral Presentations
31
The toxicity of NLPs towards plant plasma membrane Katja Pirc
1 1, Vesna Hodnik
1,2, Tina Snoj
1, Marjetka
Podobnik
1, Gregor Anderluh
11Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) /
2Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia)
Many microbial pathogens produce proteins that are toxic to the cells that they are targeting.
Nep1-like proteins (NLPs) are secreted by taxonomically nonrelated microorganisms bacteria, fungi and oomyces. Many NLPs cause necrotic lesions of plant tissue and facilitate eudicot plant infection, but are not active against monocots. NLPs are structurally related to pore-forming actinoporins and were shown to induce eudicot plant plasma membrane leakage1. Recently, we identified glycosylinositol phosphorylceramides (GIPCs), most membrane2. Cytotoxic NLP from oomycete Pythium aphanidermatum (NLPPya) undergoes structural changes after binding to the terminal hexose moiety of GIPC. We suggested a model of initial steps of NLPPya - membrane interaction and proposed why the membranes of monocots and eudicots differ in NLP sensitivity2. However, it remains unclear how the NLPs lyse cell membrane - either via pore-forming mechanism or other type of membrane integrity disruption.
The data obtained by liposome sedimentation assay, dynamic light scattering, native triptophane fluorescence and giant unilamellar vesicles (GUVs) imaging contribute to our understanding of the nature and mechanism of NLP - membrane association.
1Ottmann et al., 2009, PNAS 106, 25, 10359-10364. / 2 et al., 2017, Science 358, 1431-1434.
Orthocaspases: prokaryotic caspase homologues are regulating the toxin-antitoxin systems in cyanobacteria
, Aleksandra Uzar, Marko Dolinar
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana (Slovenia)
In Metazoa, the main actors of apoptosis are proteases termed caspases. Unexpectedly, genes coding for structurally homologous proteins were identified also in prokaryotes and are especially abundant in cyanobacteria. We have been the first to biochemically characterize these proteins and have due to their catalytic properties and evolutionary early emergence termed them orthocaspases.
We recently discovered that in the genome of the cyanobacterium Microcystis aeruginosa PCC 7806 orthocaspase MaOC1 is encoded next to putative toxin-antitoxin (TA) systems (Figure). These systems are genetic modules consisting of two co-produced genes: a relatively stable toxin that inhibits cell growth and a more labile, degradation-prone antitoxin inhibiting the lethal toxin action. They were first reported as addiction systems responsible for plasmid maintenance of cells harbouring low-copy plasmids. In plasmid-free daughter cells, the action of toxin can no longer be counteracted due to degradation of antitoxin, leading to cell death.
In the genome of M. aeruginosa orthocaspase gene is followed by TA systems, which belong to superfamilies ParE/RelE and VapB/VapC, respectively. Moreover, we observed that the first antitoxin downstream of the orthocaspase gene contains several Arg-Arg motives, which are preferred cleavage sites for MaOC1. We expressed the two antitoxins and could show that MaOC1 specifically cleaves one of them. This leads to conclusion that the cell death related enzyme is capable of inactivating antitoxins, thus releasing the toxic potential of the cognate toxins.
In a cyanobacterium Microcystis aeruginosa PCC 7806 the orthocaspase MaOC1 gene is followed by two toxin-antitoxin pairs. The MaOC1 orthocaspase specifically cleaves only the neighbouring antitoxin protein.
33
Triglyceride-based nanodroplets and their interactions with lipid-
1
, Vesna Hodnik
1, Halil I. Okur
2, Simona Sitar
3, Magda -
4, Ksenija Kogej
5 1, Sylvie Roke
2, Ema
3 1
1Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia) / 2Laboratory for Fundamental BioPhotonics, Institute of Bio-Engineering, School of Engineering, École Polytechnique
Fédérale de Lausanne (Switzerland) / 3Department of Polymer Chemistry and Technology, National Institute of Chemistry (Slovenia) / 4National Institute of Biology (Slovenia) / 5Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana
(Slovenia)
membrane surfaces and embedded nonpolar cores is important in studies of physiological processes and their pathologies and is applicable to nanotechnologies. In particular, rapidly systems to overcome in vivo complexity and variability. In this study, we present a protocol for the preparation of kinetically stable nanoemulsions with nanodroplets composed of sphingomyelin (SM) and cholesterol (Chol), as amphiphilic surfactants, and trioleoylglycerol (TOG), at various molar ratios. To prepare stable SM/Chol-coated monodisperse lipid evaporation method and combined it with ultrasonication. Lipid composition, ζ-potential, gyration and hydrodynamic radius, shape, and temporal stability of the LDs were characterized and compared to extruded SM/Chol large unilamellar vesicles (LUVs). LDs and LUVs with theoretical SM/Chol/TOG molar ratios of 1/ 1/4.7 and 4/1/11.7 were further investigated for the orientational order of their interfacial water molecules using a second harmonic scattering technique, and for interactions with the SM-binding and Chol-binding pore-forming toxins equinatoxin II and perfringolysin O, respectively. The surface characteristics and binding of these proteins to the LDs SM/Chol monolayers were similar to those for the SM/Chol bilayers of the LUVs and SM/Chol Langmuir monolayers, in terms of their surface structures. We propose that such SM/
Chol/TOG nanoparticles with the required lipid compositions can serve as experimental models for monolayer membrane to provide a system that imitates the natural lipid droplets.
Elucidating the mechanisms of lipid membrane interactions with various active substances
Mojca Mally, Janja Majhenc, Bojan Vrhovec Hartman, S Svetina, Jure Derganc
Institute of Biophysics, Faculty of Medicine, University of Ljubljana (Slovenia)
The studies on the interactions between lipid vesicle membranes and various substances are presented. Lipid bilayers are a fundamental part of cell membranes and thus the basis of each cell and cellular organelles. To understand the lipid bilayer responses to various substances we have developed a methodology of micropipetting, which allows for the transfer of spherical lipid vesicles into the solution of a substance at a certain concentration. The method of microfluidics has been developed to deal with flaccid vesicles, since in the microfluidic chamber the active ingredients are approaching the vesicles only through diffusion and there are no hydrodynamic flows. Both methods enable monitoring and the analysis of individual vesicles continuously (as opposed to analyzing a larger number of vesicles in samples, with capturing data at certain times). Thus, we have obtained insights into certain details of the interactions, which would otherwise remain hidden. Based on the results of studying different active ingredients from peptides and proteins to fatty acids and polysaccharides, in each case we designed a theoretical model with the simplest explanation of the observed interaction results. The model predicted the vesicle responses for a wide range of the active substance concentrations. A comparison between the results of the experiments and the forecasts of the model also allowed for a quantitative interaction analysis.
Shape transfarmations of a lipid vesicle due to the interaction of the membrane with bacterial lipopolysaccharide (LPS) in microfluidic diffusion chamber. / Mally et al., 2017, RCS Adv 7, 36506- 36515.
35
The venom of the white gorgonian Eunicella singularis:
novel insights into the evolution of Anthozoan toxins Maria Vittoria Modica
1,2, Marco Gerdol
3, Samuele Greco
3, Sebastien
Dutertre
1Institute of Biomolecules Max Mousseron, University of Montpellier(France) / 2Stazione Zoologica Anton Dohrn (Italy) / 3University of Trieste (Italy)
Toxinological research on Cnidaria venom has been mostly focused on sea anemones, Anthozoans belonging to the subclass Hexacorallia. Conversely, the only biochemical data available on Octocorallia or soft corals tackle their small molecule diversity, completely neglecting venom proteins and peptides. This lack of information hampers a full understanding of the evolutionary history of Anthozoa venom toxins.
To fill this gap, we used a combined transcriptomic-proteomic approach to investigate venom components in the white gorgonian Eunicella singularis, a Mediterranean soft coral inhabiting rocky bottoms at 20-30 m depth. Each colony is made up of multiple small polyps equipped with nematocyst-rich tentacles that are used to feed on zooplankton, and to deter potential predators.
Matching MS/MS data of chemically extracted nematocyst content against a whole body assembled transcriptome, we were able to identify multiple components in E. singularis venom including potential cytolytic, hemostasis impairing and neurotoxic molecules. While some of them appear to correspond to new domains and structural folds, possibly implying novel activities, others belong to well-established cnidarian toxin families, such as small cysteine rich peptides (SCRPs) and ShK toxins (ShKT). Along with the conservation of functionally important residues, we highlighted some peculiarities of E. singularis members of these protein families, such as domain duplications and additional disulfide bonds.
Phylogenetic analyses allowed us to shed a new light into the evolutionary trajectories of conserved protein families in Anthozoan venom.
Pore-forming toxin as an operator of logic gates , Gregor Anderluh
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) Construction of logic gates on lipid membranes is a novel approach in synthetic biology, challenging our understanding of lipid membrane properties, protein characteristics and functioning of cells. We exploited the ability of the engineered toxin listeriolysin O, a cholesterol-dependent cytolysin from bacteria Listeria monocytogenes, to form pores in biological membranes and to act as a logic system operator controlled by multi-level inhibition. The engineered listeriolysin O possesses suppressed activity at higher pH values.
In this way, pH ≥ 6.5 presents one level of inhibition, whereas the other is the cleavage agent (protease, reductant etc.) of its specific small protein domain inhibitor D22, designed and selected from DARPin (designed ankyrin repeat protein) scaffold library. When low pH in a combination with one or more cleavage input signals is present (OR-AND logic gates), the output is triggered and the formation of the pore at the surface of lipid vesicles occurs. As the logic gate system is constructed on the vesicle membrane, the consequence of the output signal is the release of the desired molecule, which can be chosen from a variety of options (e.g. drugs, fluorescent molecules, enzymes, binding partners). These could include also large biomolecules up to 150 kDa, owing to the rare phenomenon of well-defined large pores formed by listeriolysin O that may reach 25-40 nm in diameter.
37
Structural characterization of the lysenin pre-pore state by hydrogen/deuterium exchange mass spectrometry
Magdalena Kulma
1, Katarzyna Kwiatkowska
21Institute of Biochemistry and Biophysics, Polish Academy ofSciences (Poland) / 2The Nencki Institute of Experimental Biology, Polish Academy of Sciences (Poland)
Lysenin is a pore-forming toxin derived from coelomic fluid of the earthworm Eisenia foetida that belongs to the aerolysin family. Lysenin upon specific binding to sphingomyelin- containing lipid membrane undergoes a series of structural changes that leads to conversion of water-soluble monomers into oligomers, resulting in formation of transmembrane β-barrel pores and cell lysis. Although the structure of soluble monomer and transmembrane pore has been described recently, the underlying structural details of oligomerization await clarification. In order to gain detailed information on the mechanisms that control conformational changes required for pore formation, we have measured hydrogen-deuterium exchange pattern of peptide bond amide protons in lysenin and its double cysteine mutant V88C/Y131C, in which disulfide bond between β6 and β10 strands arrests lysenin oligomers in the pre-pore state. In this study, the application of hydrogen-deuterium exchange analysis allowed to characterize structural dynamics of lysenin at the pre-pore state. We demonstrated increased structural stabilization of lysenin upon oligomerization in comparison to water- soluble form. Stabilization was enhanced in the POC-binding region within the C-terminal head domain, and was transmitted by the link between N and C domains to the β-strand (β7-β11). Based on these results we suggest that stabilization of these regions drives conformational changes leading to pore formation.
Listeriolysin O tryptophans are involved in cholesterol-rich membrane binding and toxin's hemolytic activity Mirijam Kozorog
1, Marc-Antoine Sani
2, Martina
1, Janez
Plavec
1,3,4, Frances Separovic
2, Gregor Anderluh
11Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) /
2School of Chemistry, Bio21 Institute, University of Melbourne (Australia) / 3Slovenian NMR Centre, National Institute of Chemistry (Slovenia) / 5EN-FIST Centre of Excellence (Slovenia) / 4Faculty of
Chemistry and Chemical Technology, University of Ljubljana (Slovenia)
Listeria monocytogenes is human and animal pathogen bacterium that causes gastroenteritis, miscarriages and infections of the central nervous system in immunocompromised individuals.
Its main virulence factor is listeriolysin O (LLO), a pore-forming toxin that enables the bacterial escape from acidic phagosome, bacterial spread to neighbouring cells and it also contributes to bacterial pathogenicity. Listeriolysin O belongs to cholesterol-dependent cytolysins (CDC), which are known to form pores exclusively on cholesterol-rich membranes.
The binding occurs via domain 4 (D4), but the process has not been explained in details yet.
Since previous studies suggested that tryptophan aminoacid residues are involved in the membrane binding, we addressed this question by expressing recombinant listeriolysin O with all seven tryptophans labelled with 19F. With solution and solid-state NMR spectroscopy we recorded the 19F-NMR spectra of the labelled protein in solution and, separately, when it was bound to cholesterol-rich multilamellar vesicles. We also expressed single W F listeriolysin O mutants for NMR spectra peaks assignation and compared their hemolytic activity with the wild-type protein. The results show 3 tryptophan residues play a role in the studied process; W189 and W489 being involved in pore-forming proces and W512 interacting with the membrane1.
1Kozorog et al., 2018, Sci Rep 8, 6894.
Abstracts - Posters
41
APS8 induces apoptosis of the NSCLC model cell line by interfering with the lung cancer signalling pathways
Sabina Berne
1, Simona Kranjc
2 2,3, Tom Turk
41Department of Agronomy, Biotechnical faculty, University of Ljubljana (Slovenia) / 2Institute of Oncology Ljubljana (Slovenia) / 3Faculty for Health Sciences, University of Primorska (Slovenia) /
4Department of Biology, Biotechnical faculty, University of Ljubljana (Slovenia)
The poly-APS synthetic analogue, APS8, is a potent antagonist of α7 nicotinic acetylcholine receptors (nAChRs), which are emerging as a potential target for lung cancer therapy.
Recently, the therapeutic value of APS8 has been demonstrated in human adenocarcinoma xenograft models. APS8 was not toxic to mice up to 5 mg/kg i.v. and induced no significant histological changes but has significantly delayed tumour growth and generally prevented regrowth of tumours.
Using Kinex Antibody Microarrays, we examined the underlying molecular mechanisms of APS8-induced apoptosis of a model NSCLC cell line. Following the exposure of the A549 cells to 500 nM APS8 for different time intervals (5, 15, 30, 60 min and 24 h), cell lysate proteins were fluorescently labelled, mounted onto the microarrays and changes in the protein expression or phosphorylation were determined with a laser array scanner. Background- corrected, log2 transformed and globally normalized intensity data were visualized as protein interaction networks in the Cytoscape platform.
APS8 treatment induced intrinsic apoptosis signalling pathway in response to DNA damage in A549 cells by downregulating MAPK and STAT signalling cascades, calreticulin and ERBB2, and upregulating cyclin dependent kinases (CDK1, CDK5, CDK6 and CDK13) involved in the cell cycle control. In addition, the exposure of A549 cells to APS8 increased the protein expression of CaMK2G and CAMK4 kinases associated with calcium triggered signalling cascades. After 24 h, an increased expression of DIABLO and activation of caspases was detected. These molecular mechanisms of APS8-induced apoptosis will be further investigated and validated with immunoblotting experiments
Vesiculation of mammalian cells in response to listeriolysin O and its mutants
Apolonija Bedina Zavec
1 1, Matic Kisovec
1, Maja Jamnik
1, Veronika Kralj-
2, Gregor Anderluh
1, Marjetka Podobnik
11Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry (Slovenia) /
2Biomedical Research Group, Faculty of Health Sciences, University of Ljubljana (Slovenia)
Listeriolysin O (LLO) is a toxin from the intracellular pathogen Listeria monocytogenes, which forms pores in cholesterol-rich lipid membranes of host cells. Besides its biological relevance, LLO and its pore forming ability is also interesting for the applications in medicine and biotechnology. Two mutant proteins were generated by our group, Y406A and A318C+L334C. The single mutant Y406A is able to bound to membranes and oligomerizes similarly to the wild-type LLO (wtLLO), but in contrast to wtLLO, the final membrane insertion step of Y406A mutant is accomplished only under acidic pH. The double cysteine mutant A318C+L334C does not exhibit hemolytic activity in the oxidized state, but it bound to the lipid membranes to the same extent in both, reduced or oxidized state. The release of extracellular vesicles (EVs) and cytotoxicity were used to examine the response of mammalian cells to LLO and its mutants. Three different cell lines were used and the most appropriate cell line for our experiments was K562. Mutant Y406A was not cytotoxic at neutral pH, while at pH6 it showed almost the same cytotoxicity as wtLLO at pH7.4.
Mutant A318C+L334C in the oxidized state was about 100-fold less cytotoxic than the wtLLO, and of about 10-fold less cytotoxic than wtLLO when in the reduced state. Both mutants were thus significantly less toxic than wtLLO under physiological conditions and become toxic either under acidic conditions (Y406A) or reduction (A318C+L334C); therefore, these mutants can be used in stimuli responsive applications. The level of EV secretion was significantly increased at cytolethal concentrations of all three proteins. Vesiculation level was also increased at about 10-fold lower concentrations than cytolethal. However, at about 100-fold lower concentrations than cytolethal, the effect was reversed and cells shedding less EVs than control cells. EV shedding acts as the main clearance mechanism for LLO at cytolethal and subcytolethal concentrations in cell line K562; whereas at lower concentrations the endocytosis is probably the main mechanism to prevent pore formation.
43
Isolation and characterization of C-type lectin-like proteins (snaclecs) from Vipera ammodytes ammodytes (Vaa) venom inducing transient and reversible thrombocytopenia
of functional platelets
Mojca Dobaja Borak
1, Adrijana Leonardi
2, Miran Brvar
1, Vid Leban
1, Igor
2
1Centre for Clinical Toxicology and Pharmacology, University Medical Centre Ljubljana (Slovenia) /
2Department of (Slovenia)
Platelets play an essential role in the initial response to vascular injury. In thromboembolic diseases like myocardial infarction and ischemic stroke they participate in early stage of the pathophysiological process. There are already some antiplatelet drugs existing that are used to prevent and reverse platelet aggregation in acute coronary disease and they work as GP IIb/IIIa receptor antagonists of irreversibly activated platelets. However, in interventional angiology and cardiology, an agent is needed with a transient and reversible antithrombotic effect without impairment of primary hemostasis (e.g. platelet activation). Profound and transient thrombocytopenia of functional platelets without bleeding was observed in patients bitten by Vaa at the University Medical Centre Ljubljana and thrombocytopenia could be rapidly reversed with antivenom application. So far different components were found in Vaa venom which affects hemostasis1. Among them, non-enzymatic dimeric proteins, disintegrins and snaclecs, acted as strong inhibitors of platelet aggregation. The latter are known as efficient inhibitors of ristocetin-induced platelet agglutination suggesting the involvement of GPIb platelet receptor in the observed effect1,2 that cause thrombocytopenia in vivo. Pool of isolated Vaa snaclecs added to whole blood samples caused thrombocytopenia due to aggregates or agglutinates. In addition, a flow cytometry revealed a specific interaction between snaclecs and the platelet GPIb receptor without expression of GPIIb/IIa and P- selectin, the markers of platelet activation, what is in accordance also with the results of preliminary flow cytometry of platelets of our Vaa bitten patients. We are currently working on purification and characterization of novel snaclecs from Vaa venom in order to evaluate their interaction with specific platelet receptors and to reveal the mechanism of thrombocytopenia.
1Sajevic et al., 2013, Toxin Rev 30, 33-36. / 2Eble JA, 2019, Toxins (Basel) 11(3).
Interaction between ammodytoxin, a β-neurotoxin from the nose-horned viper venom, and neuronal mitochondria
1,2 1 2 1
1 (Slovenia) / 2Faculty of
Medicine, University of Ljubljana (Slovenia)
β-neurotoxins (β-ntxs) block neuro-muscular transmission by poisoning nerve terminals, causing flaccid paralysis1. Structurally and functionally they belong to secreted phospholipases A2 (sPLA2), a group of enzymes secreted by cells. Secreted PLA2s hydrolyse glycerophospholipids to sn-2 lysophospholipids and fatty acids. However, not all sPLA2 are toxic, they have been described from viruses to mammals, being implicated in many disease, when the activity of endogenous group IIA sPLA2 (GIIA sPLA2) is elevated and it becomes toxic for neurons, inducing damage to the mitochondria. This effect is very similar to that induced by some β-neurotoxins structurally homologous to GIIA sPLA2. While the molecular mechanism of this action of GIIA sPLA2 is not known, the β-ntxs have been better studied in this respect and might be used as tools for revealing the former. In that regard, a high affinity membrane receptor for ammodytoxin (Atx), a β-ntx from the venom of the nose- horned viper (Vipera a. ammodytes), was detected in neuronal mitochondria and identified as the subunit II of cytochrome c oxidase (CCOX-II), an essential constituent of the respiratory chain complex2. It was shown that Atx inhibits CCOX activity when incubated with isolated mitochondria. How β-ntxs translocate into mitochondria, however, remains to be investigated. To study this and similar processes we have labelled an enzymatically inactive mutant of Atx with 5 nm NHS-activated gold nanoparticles. We are using this molecular tool for localization studies by electron microscopy, investigating internalization of Atx into a neuron-like model cell line and its translocation into mitochondria. Altogether, the results suggest the explanation of the mechanism by which β-ntxs hinder the production of ATP in poisoned nerve endings and open an important direction of study to advance the understanding of the mitochondrial function and dysfunction of endogenous GIIA sPLA2.
1 et al., 2014, Toxicon 89, 9. / 2 et al., 2019, Sci Rep 9, 283.
