Seminars

Forthcoming Programme Group Seminars

Seminars in 2023-2024

14. 11. 2024 Mojca Dobaja Borak (PhD student at MF and JSI): Isolation and characterization of proteins from long-nosed viper (Vipera ammodytes ammodytes) venom that induce transient and reversible thrombocytopenia of functional platelets

Abstract

In Europe, the nose-horned viper (Vipera ammodytes ammodytes, Vaa) is the most poisonous snake, with severe thrombocytopenia as the most common laboratory finding upon envenomation with its venom. No agonists of platelet aggregation have been detected in Vaa venom so far, so we hypothesized that snake C-type lectin-like proteins of the venom (Vaa-snaclecs) could be responsible for this pathological effect. Our study aimed to purify Vaa-snaclecs in order to investigate their ability to bind platelet receptors and thus trigger their agglutination. In the biochemical part of our research, we isolated Vaa-snaclecs from the crude venom by a combination of different liquid chromatography techniques and confirmed their purity by LC-ESI-MS/MS. For the most abundant one, an acidic and non-glycosylated 30 kDa Vaa-snaclec-3/2, we tested platelet agglutination/aggregation ability using turbidometry and binding to platelet receptors and platelet activation by flow cytometry. It bound to the platelet GPIb receptor and caused a reversible platelet agglutination, inducing a drop of the platelet count, without activating these cells. Structurally, Vaa-snaclec-3/2 is a heterodimer consisting of the α-subunit 3 and β-subunit 2. Using thromboelastometry, we evaluated platelet functionality in patients envenomed by the Vaa venom expressing thrombocytopenia, before and after antivenom therapy with F(ab')₂ fragments against the whole Vaa venom. Antivenom treatment successfully reversed thrombocytopenia in all patients within one hour. We also conducted an in vivo study in a mouse model of arterial thrombosis that showed Vaa-snaclec-3/2 causes thrombocytopenia and protects the experimental animals from arterial occlusion. The reversible thrombocytopenia of functional platelets by Vaa-snaclec-3/2, which inhibits thrombus formation, could be beneficial in interventional procedures that require only a temporary reversal of platelet adhesion/aggregation, such as balloon dilation, stent implantation, and embolus aspiration.

The seminar will be held online on Thursday, November 14, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

17. 10. 2024 Gašper Žun (PhD student at JSI): Outcomes of the NHEJ-induced repair of CRISPR-Cas9 generated DSBs in the yeast Saccharomyces cerevisiae

Abstract

Endonuclease Cas9 creates blunt ended double-strand breaks (DSBs) that must be repaired either by homology-directed repair (HDR) or non-homologous end joining (NHEJ) for the cell to survive. In the yeast S. cerevisiae, the HDR is effective to the degree that some even argue that NHEJ does not operate at all. In fact, that contradiction – compared to most other organisms where NHEJ is more active than HDR – led yeast to become a model organism in genomics. However, yeast possess all the components of the NHEJ process, yet their efficiency is low. To locally induce the NHEJ repair, we fused each participating component of the NHEJ machinery directly to the Cas9. We reasoned that after the DSB generation the repair at the hotspot would be taken over by the nearby NHEJ component to induce the process. The outcomes of DSB repair in these strains were phenotypically assessed and evaluated by in-depth sequencing of the sites of repair. The results were compared between different genetic backgrounds of genes involved in DNA repair mechanisms, and between three different loci. Generally, the MRX complex induced deletions, whereas insertions were induced by the error-prone Pol4 DNA-polymerase. A significant portion of the outcomes could be associated to the NHEJ-components mechanism and nearby sequence dependence. Consequently, discoveries that will be presented at some points even disprove and redefine textbook facts about NHEJ-generated outcomes.

The seminar will be held online on Thursday, October 17, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

13. 6. 2024 Luka Žeželj (PhD student at BF UL): Aegerolysins as markers of early apoptosis

Abstract

Aegerolysins are »15-kDa proteins found predominantly in bacteria and fungi. Their common feature is the ability to interact with membranes that contain the invertebrate-specific membrane sphingolipid ceramide phosphoethanolamine. Most of the aegerolysins from edible oyster mushrooms (Pleurotus sp.) also interact specifically with the mammalian membrane sphingolipid sphingomyelin (SM), which is found in a complex with cholesterol. These aegerolysins can be used as universal markers to detect membrane rafts and monitor their dynamics in mammalian cel membranes. Furthermore, in the presence of »60-kDa partner proteins PlyB or PulB, which are also produced by Pleurotus mushrooms and bear a MACPF domain, aegerolysins can form multimeric bicomponent complexes that create transmembrane pores in mammalian or in insect cells. This pore formation causes cell death.

Aegerolysin erylysin A (EryA) from the king oyster mushroom (P. eryngii) is the only aegerolysin that does not bind to the SM/cholesterol combination, but only to the membrane ceramide phosphoethanolamine. In addition, the recombinant fluorescent version of EryA (EryA-mCherry) specifically senses and binds to cardiolipin, which is a glycerophospholipid specific for bacteria and for the inner mitochondrial membrane.

Membrane lipid rearrangement is a process that can be observed during the process of apoptosis, which is an evolutionarily conserved and universally regulated cell death pathway of (higher) metazoans. To date, the best described apoptotic signal is the exposure of the membrane glycerophospholipid phosphatidylserine in the outer layer of the plasmalemma of the apoptotic cell. It has been shown that during the process of apoptosis in mammalian cells, cardiolipin is also exposed on the surface of the membrane, as proapoptotic stimuli damage the mitochondria and stimulate the translocation of cardiolipin from the inner through the outer mitochondrial membrane to the plasmalemma. Translocation of cardiolipin occurs very early during apoptosis – even before changes in mitochondrial membrane potential or phosphatidylserine exposure occur. As part of the doctoral thesis, we will evaluate the binding of fluorescently labelled EryA to artificial lipid membranes containing cardiolipin, and evaluate its potential as a marker of early apoptosis in mammalian cells.

The seminar will be held online on Thursday, June 13, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

13.6. 2024 Tadeja Bele (PhD student at JSI and BF UL): Antagonists of α7-nicotinic acetylcholine receptor and their potential therapeutic effects on lung cancer cells

Abstract

Receptors and their signalling cascades play a crucial role in regulating cellular processes, and their dysregulation leads often to disease development. In particular, elevated expression of nicotinic acetylcholine receptors (nAChRs) has been observed in various types of cancer, including lung cancer. The binding of ACh or other agonists, such as nicotine and its derivatives, to these receptors has been linked to uncontrolled cell division, prevention of apoptosis and induction of angiogenesis, ultimately supporting tumour growth and metastasis. In contrast, antagonists of nAChRs have shown opposite effects on cells, suggesting their potential value in cancer therapy. Vulfius et al. (2014) showed that secreted phospholipases A₂ (sPLA₂s) from Elapidae and Viperidae snake venoms act as antagonists of nAChRs by supressing ACh-elicited ion currents. This effect was independent of phospholipase activity. Several human orthologues of snake venom sPLA₂s exist and we were interested whether or not their physiological function is also regulation of nAChR activity. We prepared recombinant human sPLA₂s, group V (hGV) and X (hGX), as well as their single point active site mutants devoid of enzymatic activity. Electrophysiological assessment of their effects on α7 and adult muscle type nAChRs expressed in Xenopus laevis oocytes were performed. All four human sPLA₂ molecules inhibited ion current that was triggered by ACh through α7 nAChR. Interestingly, the analysis revealed selectivity of enzymatically inactive hGV for α7 nAChRs. Furthermore, we analysed the expression of α7 nAChR and dup-α7 nAChR, a truncated version of the α7 subunit that is lacking 146 residues of the ligand binding domain, in various lung cancer cell lines as well as in BEAS-2B cell line, a human non-tumorigenic lung epithelial cell line. H23 adenocarcinoma cell line proved to be the best model, for further analysis. Currently, we are testing the effects of hGV and its mutant on cell proliferation and their ability to prevent the pro-cancer effects of nAChR agonists.

16. 5. 2024 Kity Požek (PhD student at JSI): Therapeutic potential of the nose-horned viper’s haemostatically active venom proteins

Abstract

Cardiovascular diseases (CVD) are a leading cause of mortality worldwide and account for around a third of all global deaths. Current therapies for CVD, using antithrombotics, anticoagulants and thrombolytics, often lead to undesirable side effects, particularly bleeding. Snake venoms, rich in pharmacologically active compounds, offer a promising avenue for the development of more effective and safer therapeutic alternatives. Our study explores the therapeutic potential of haemostatically active proteins in the venom of the nose-horned viper (Vipera a. ammodytes; Vaa). The aim of the study is to isolate and characterize new proteins from Vaa venom that have significant effects on the blood or cardiovascular system. A key focus of our research is on anticoagulant protein VaaSPH-1, which was previously identified in the viper venom. We are establishing an effective procedure to recombinantly produce this glycoprotein and its mutants in the HEK293F cell line to advance our knowledge about its interaction with its most important biological target, the blood coagulation factor VIIIa (FVIIIa) in the intrinsic tenase complex. The precise structural description of the interaction between FVIIIa and VaaSPH-1 will be the basis for development of low molecular mass anticoagulants–peptides or peptidomimetics–for safer treatment of venous thromboembolism

The seminar will be held online on Thursday, May 16, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

16. 5. 2024 Anja Pavlin (PhD, researcher at the Biotechnical Faculty): Autoregulation ensures vertical transmission of the linear plasmid prophage GIL01

Abstract

Betatectiviruses are unusual prophages consisting of linear extrachromosomal genomes that lack the plasmid component. It remains unclear how plasmidial betatectiviruses are maintained in low-copy numbers in host cells and how they are vertically transmitted. Phage GIL01 is a model betatectivirus that infects the mosquito pathogen Bacillus thuringiensis serovar israelensis. Previous studies have identified two closely spaced promoters, P1 and P2, responsible for the expression of GIL01 genes required for prophage replication and the switch from the lysogenic to lytic cycle. Here, we report that the GIL01-encoded, 58-amino acid long gp1 protein forms a large nucleoprotein complex that represses its transcription from the strong promoter P2. Notably, ectopic expression of gp1 resulted in the loss of GIL01 in exponential cultures and immunized cells against infection with GIL01, indicating that gp1 plays a repressive role in the phage cycle. This finding is consistent with mutations in gp1 committing GIL01 to the lytic cycle; we showed that maintenance of this phage variant in the bacterial population is affected by the accumulation of deletions in the P1-P2 region. The fact that gp1 is conserved across most sequenced betatectiviruses suggests that the regulatory mechanism of gp1 that controls prophage maintenance is widespread among these bacteriophages.

18. 4. 2024 Neža Škofljanc (PhD student at JSI): Species-wide variation in azole drug resistance in model organism Saccharomyces cerevisiae

Abstract

Invasive fungal infections are posing an alarming threat to public health system, causing an estimate of 1.5 million human deaths per year. Due to an increasing number of immunocompromised patients, which are predominantly affected by fungal infections, the emergence of drug resistant pathogens is on the rise. To better understand the genetic determinants causing antifungal drug resistance we inspected this trait in the model organism Saccharomyces cerevisiae on a species-wide scale. S. cerevisiae is found in a variety of environmental niches, including the human body as an opportunistic pathogen. Several studies have shown conserved fungal virulence factors as observed in more common fungal pathogens, such as Candida, Cryptococcus and Aspergillus. We tested a large cohort of 1011 natural S. cerevisiae strains in the presence of different antifungals. The phenotypic data obtained allowed us to perform a genome-wide association study, which revealed 420 genetic variants associated with the regulation of antifungal drug resistance. To experimentally validate the phenotypic effect of discovered variants, we chose some of the most interesting ones and engineered them into different S. cerevisiae genetic backgrounds. Those variants were found in four different genes – HSP82, AIM29, PIS1 and FKS1 – all involved in general cell stability and stress response pathways, rather than more specific, already known drug response related genes. This suggests that natural isolates primarily exert resistance through different mechanisms, compared to the isolates that are under more direct evolutionary pressure, such as clinical isolates. With the use of CRISPR-Cas9 technology we engineered the chosen variants into one laboratory and one natural S. cerevisiae strain and tested their effect in different antifungal drugs. All the variants tested, in at least one genetic background, caused a change in drug sensitivity between the parent and engineered strains. However, we observed a significant difference in the effect of variants, depending on the genetic background in which they were present. The impact of genetic background on complex trait expression is a commonly observed phenomenon, yet difficult to dissect. To gain better understanding of it, we are now further developing a project in which we aim to explore the extent to which genetic background affects complex traits.

The seminar will be held online on Thursday, April 18, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

18. 4. 2024 Mia Žganjar (PhD student at JSI): Advancing oleochemical bioproduction: Insights from Yarrowia lipolytica mRNA-Seq analysis

Abstract

Commercial production of microbial oils has not yet reached an economically feasible turning point. The carboxylate platform was suggested as a promising strategy, converting volatile short-chain fatty acids (SCFA) into high-value oleochemicals via oleaginous yeasts. Through an extensive hight-througput screening of 1,434 diverse yeasts, Yarrowia lipolytica EXF-17398 emerged as a suitable strain capable of efficient SCFA conversion to microbial oils.

To gain insights into the biological mechanisms driving SCFA uptake and metabolic processing, mRNA-Seq and multi-condition differential gene expression analysis were conducted, comparing the expression patterns between glucose, acetic acid and propionic acid utilisation. Functional analysis entailed over-representation analysis and gene set enrichment analysis. Furthermore, a genome-scale metabolic model was created by integrating expression data to the media adjusted iYLI647 model with the cost optimization reaction dependency assessment model extraction method.

Detailed transcriptomic insights into Y. lipolytica EXF-17398 strain revealed its unique upregulation mechanisms in ion homeostasis and the transport mechanisms. Predicted growth rates of created models were benchmarked to experimentally obtained data and could accurately simulate the conversion processes from SCFAs to microbial oils, offering insights into potential metabolic engineering targets for optimizing oleochemical yields. Our findings bring us closer to understanding the molecular pathways driving efficient SCFA conversion in Y. lipolytica, potentially enhancing microbial oil production through targeted engineering.

14. 3. 2024 Maja Hostnik (PhD student at BF UL): Influence of GIL01 phage on Bacillus thuringiensis sporulation

Abstract

GIL01 infects endospore-forming bacterium Bacillus thuringiensis serovar israelensis which is used as an efficient biopesticide worldwide. Following infection, GIL01 prophage enters lysogenic cycle and resides within the host as autonomous linear plasmid without integrating into the chromosome. Lysogenic phages generally establish a stable relationship with their host bacteria and can in some cases transfer novel properties, like virulence traits or enhanced survival capabilities, to the bacterial host. Since GIL01 genome is a small extracellular replicon, we assumed its genome could be incorporated inside of a bacterial endospore. Bacterial endospores can serve as phage genome protection shells against various environmental factors and can therefore enhance their survival in unfavourable environments.

The aim of our work was to evaluate potential influence of GIL01 on sporulation of its host B. thuringiensis serovar israelensis. For this purpose, a B. thuringiensis serovar israelensis plasmid cured (nonlysogenic) strain and an entomopathogenic B. thuringiensis serovar israelensis strain isolated from soil in Brazil were used. Sporulation rates of strains harbouring or not the GIL01 prophage were evaluated. The results show that GIL01 lysogeny has a significant influence on the sporulation rate in plasmid-cured B. thuringiensis strain, while the phage influence in the entomopathogenic strain is minor. We also confirmed that most of the spores carry GIL01 genome. Currently, we are investigating mechanisms underlying GIL01 influence on B. thuringiensis sporulation.

The seminar will be held online on Thursday, March 14, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

15. 2. 2024 Špela Koren (PhD student at JSI): A dynamic interplay of cytosolic and lysosomal lipid droplet breakdown pathways in starved breast cancer cells

Abstract

Lipid droplets (LDs) are cytosolic fat storage organelles involved in various cellular stress responses. Starving cells typically employ cytosolic neutral lipases for LD breakdown, but nutrient deprivation also triggers upregulation of various forms of autophagy, including the selective breakdown of LDs via lipophagy. Here, we aimed to investigate the interplay between autophagy/lipophagy and the regulation of LD turnover in starving breast cancer cells. Despite recent progress in the understanding of the mechanisms of LD breakdown, the question of when and why cells employ different pathways of LD breakdown still remains unanswered. Our recent findings reveal a continuous cycle of LD formation and breakdown during prolonged serum starvation. While diacylglycerol acyltransferase (DGAT)-driven LD accumulation occurs even after several days of serum starvation, autophagy does not contribute to LD biogenesis under these conditions. Instead, autophagy/lipophagy facilitates LD breakdown and is active alongside lipolysis. Depletion of adipose triglyceride lipase (ATGL), the initiator of intracellular lipolysis, reduces LD breakdown, prevents fatty acid transfer from LDs to mitochondria, and impairs lipophagy. Suppression of autophagy/lipophagy by silencing essential autophagy genes during prolonged starvation results in a compensatory LD breakdown through ATGL-mediated lipolysis leading to LD depletion. These findings suggest a dominant role of ATGL in LD turnover through both lipolysis and lipophagy. However, even when both ATGL and autophagy/lipophagy are inhibited, cancer cells still break down LDs through an autophagy-independent lysosomal mechanism mediated by lysosomal acid lipase (LAL). Overall, our findings demonstrate a dynamic interplay between cytosolic lipolysis and both autophagy-dependent and autophagy-independent lysosomal LD breakdown mechanisms. Our ongoing objective is to clarify how these mechanistically distinct pathways of LD breakdown contribute to membrane homeostasis, lipid signalling, energy metabolism and overall protection of cancer cells against nutrient stress.

The seminar will be held online on Thursday, February 15, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

15. 2. 2024 Larisa Lara Popošek (PhD student at BF UL): The quest for new lipid-binding proteins and biotechnological applications

Abstract

So far known aegerolysins are characterized by high affinity for the lipid receptor ceramide phosphoethanolamine (CPE), which is characteristic for some insects. In addition, some of these aegerolysins less specifically bind to the mixture of sphingomyelin (SM) and cholesterol (Hol), which is characteristic lipid mixture in vertebrate membranes. Fluorescently labeled aegerolysins can be used to study these lipids, in the similar manner as antibodies are used to study the localization or presence of proteins in the cells. Although aegerolysins are non-toxic, they may, in the presence of a protein with the MACPF domain, become part of a cytolytic complex that perforates the membrane of those cells that contain the lipid receptor recognized by aegerolysin. Aegerolysins are therefore biocomponent cytolysins - component A (aegerolysin) recognizes the lipid target, and component B (protein partner with the MACPF domain) further allows the formation of pores in the target membrane.

The four aegerolysins and their MACPF protein partners are coded by basidoimicetes Heterobasidion irregulare, Trametes versicolor, Lepista nuda and Mucidula mucida. We have tried obtain all these proteins in recombinant and the first experiments have been performed. You will therefore find out in the seminar: (i) whether these novel aegerolysins also recognize lipid receptors, (ii) whether do these aegerolysins in combination with MACPF protein partners show any hemolytic activity and whether they are (iii) insecticidal.

18. 1. 2024 Tim Nograšek (MSc student at FCCT UL): Transport of LINE1 retrotransposons into the nucleus by karyopherins

Abstract

My master’s research is on nuclear import of ORF1p which is encoded in a retrotransposon LINE1 (long interspersed nuclear element 1). LINE1s are mobile DNA elements that represent as much as 17% of a human genome. LINE1 is an autonomous element that encodes two proteins ORF1p and ORF2p. Both are required for successful retrotransposition of their own RNA to a new location in the genome. After proteins are synthesised in the cytoplasm, ORF1p, ORF2p and mRNA assemble LINE1 ribonucleoprotein (RNP) complex that is transported into the nucleus. Some studies have shown that retrotransposition mostly takes place in dividing cells and on the other side, the effect of retrotransposition on somatic cells does not have big impact because they divide rarely. The requirement for cell divisions may represent a mechanism that limits L1 retrotransposition and prevents genomic instability in somatic tissues. But still some other articles show that LINE1 RNP complex needs to be transported through the nuclear membrane in different nondividing or at some point cell cycle arrested cells. The most common mechanism of importing proteins in nuclei through nuclear pore complex is with karyopherins alpha and beta. Karyopherins recognise different nuclear localisation sequences (NLS), but LINE1 does not have a typical NLS. Another way is a noncanonical way, where karyopherin alpha probably binds to basic amino acids in ORF1p.
The aim of my thesis is to determine the nuclear import of LINE1 ORF1p using nuclear import assay (NIA). In this method, the cytoplasm of mammalian cells is removed, and with the addition of reticulocyte cytoplasm and isolated protein ORF1p, the protein may be transferred to the nucleus. The method can also determine the exact composition of the complex of nuclear receptors and the cargo. We expressed in and isolated all 7 karyopherins α, karyopherin β, transportin and ORF1p from E. coli using GST tag. We got approximately 2-6 mg of protein from 1 L of bacterial culture. We are still optimizing the protocol for NIA to get unambiguous results. We have also tried to show interactions of ORF1p with karyopherins with BioID experiments and co-immunoprecipitation.

The seminar will be held online on Thursday, January 18, starting at 13:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

18. 1. 2024 Marija Kisilak (PhD student at FCCT UL): MLKL – The Phantom Menace: Oligomerization and membrane interaction

Abstract

Necroptosis is a form of regulated cell death that is triggered when apoptosis is inhibited, and leads to necrotic cell death. The main players in necroptosis are the kinases RIPK1 and RIPK3 and a pseudokinase protein called MLKL (mixed lineage kinase domain like pseudokinase). MLKL plays a central role in the execution of necroptosis and is involved in the membrane disruption that occurs during necroptosis. Understanding the molecular mechanisms underlying MLKL effector function is essential for elucidating the intricate processes of necroptosis. To better understand the oligomerization of MLKL and its interaction with the membrane, we expressed several truncated and full-length variants of the protein. Using size-exclusion chromatography and RALS/LALS, we observed the tendency of these variants to oligomerize. To gain insight into the oligomeric state of MLKL, we employed cross-linking experiments. Our data revealed the presence of MLKL dimers, trimers, tetramers and higher-order oligomers, supporting the hypothesis that oligomerization is a crucial step in MLKL-mediated membrane disruption. When MLKL was added to POPC-POPE-cardiolipin containing liposomes, significant liposome leakage was observed, demonstrating that MLKL is essential for membrane disruption. To further investigate the functional significance of MLKL in membrane disruption, we generated stable inducible FlpIn HEK293T cell lines expressing different MLKL constructs. This allowed us to examine the properties of MLKL variants and their effect on cell death. Using confocal fluorescence imaging of fixed cells and functional assays, we observed that MLKL expression resulted in membrane disruption, consistent with its role in necroptosis. Furthermore, we investigated the interaction between MLKL and a newly developed nanobody and explored the formation of MLKL oligomers and their involvement in membrane disruption. To study the interaction between MLKL and the nanobody, we performed in vitro experiments using recombinant variants of MLKL and the nanobody. Our results showed a specific interaction between MLKL and the nanobody, suggesting its potential as a valuable tool for modulating MLKL activity in various biological contexts. Further investigations into the structural properties and dynamics of MLKL oligomers will contribute to a comprehensive understanding of MLKL effector function and may provide potential therapeutic targets for diseases involving dysregulated cell death pathways.

14. 12. 2023 Leja Perne (PhD student at JSI): Lipid droplets and the control of cell fat(e)?

Abstract

Lipid droplets (LDs) are ubiquitous fat storage organelles. Their biogenesis and breakdown is dynamically altered in response to various environmental stress conditions. During stress, LDs manage the trafficking, storage and use of fatty acids for a variety of purposes in the cell. Membrane phospholipids containing polyunsaturated fatty acids (PUFAs) are highly vulnerable to lipid peroxidation, which if left uncontrolled leads to ferroptotic cell death. Our previous unpublished work revealed that LDs are involved in the regulation of ferroptosis sensitivity in the highly tumourigenic and metastatic MDA-MB-231 breast cancer cells. Our most recent investigations have shown that LD biogenesis is activated during ferroptosis to sequester membrane-derived PUFAs into neutral lipids to protect them from oxidation. We next asked if the breakdown of these ferroptosis-induced and PUFA-enriched LDs can also modulate ferroptosis. Targeting the main mechanisms of LD degradation, lipolysis and lipophagy, with pharmacological agents and siRNA knockdowns revealed a differential role for lipolysis vs. lipophagy in the regulation of ferroptosis. Our results suggest that understanding the molecular mechanisms of LD-mediated control of membrane composition, oxidation and ferroptosis may have important implications for the development of new approaches to promote ferroptosis in cancer.

The seminar will be held online on Thursday, December 14, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

16. 11. 2023 Eva Jarc Jovičić (PhD, researcher at JSI): The mobilization of fat stores from lipid droplets regulates metabolic and energy demands of starving cells

Abstract

Lipid droplets (LDs) are highly dynamic organelles, alternating between periods of growth and degradation. These processes essentially regulate metabolic and energy demands of cells under conditions of nutrient stress caused by lipid overload-induced lipotoxicity or limited nutrient availability. Starving cells typically employ cytosolic neutral lipases for LD breakdown, but nutrient deprivation also upregulates various forms of autophagy, including the selective breakdown of LDs via lipophagy. Despite recent advances on the mechanisms of LD breakdown as well as their functions, the fundamental question why cells employ distinct pathways of LD breakdown for supporting their signalling and metabolism and how these pathways are regulated remains to be answered. Our recent results suggest that a dynamic, context-dependent crosstalk exists between lipolysis and lipophagy in starving breast cancer cells. Furthermore, cancer cells with a combined inhibition of lipolysis and lipophagy were still able to breakdown LDs via autophagy-independent lysosomal mechanisms. In the seminar, we will present how these mechanistically distinct pathways of LD breakdown support membrane homeostasis, lipid signalling and energy metabolism in starving cancer cells.

The seminar will be held on Thursday, November 16, starting at 14:00 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

19. 10. 2023 Mojca Dobaja Borak (PhD student at MF and JSI): Reversible and transient thrombocytopenia induced by snake C-type lectin-like proteins (snaclecs) from the nose-horned viper venom

Abstract

In Slovenia, the nose-horned viper (Vipera a. ammodytes, Vaa) is medically the most important snake. Its venomous bite can result in severe thrombocytopenia. No agonists of platelet aggregation have been detected in Vaa venom so far, so we hypothesized that snake C-type lectin-like proteins of the venom (Vaa-snaclecs) could be responsible for this pathological effect. The aim of our work was therefore to purify Vaa-snaclecs in order to investigate their ability to bind platelet receptors and thus trigger their agglutination/aggregation. In the biochemical part of our study, we isolated Vaa-snaclecs from the crude venom by a combination of different liquid chromatography techniques and confirmed their purity by LC-ESI-MS/MS. For the most abundant one, an acidic and non-glycosylated 30 kDa Vaa-snaclec-3/2, we tested platelet agglutination/aggregation ability using turbidometry and binding to platelet receptors and platelet activation by flow cytometry. It bound to the platelet GPIb receptor and caused a reversible platelet agglutination, inducing a drop of the platelet count, without activating these cells. Vaa-snaclec-3/2 is a heterodimer consisting of the α-subunit 3 and β-subunit 2. Using thromboelastometry, we evaluated platelet functionality in patients envenomed by the Vaa venom expressing thrombocytopenia, before and after antivenom therapy with F(ab')2 fragments against the whole Vaa venom. Antivenom treatment resolved thrombocytopenia in all patients within one hour. Currently, we are conducting an in vivo study in a mouse model of arterial thrombosis to validate the potential of Vaa-snaclec-3/2 to prevent arterial occlusion and to determine its potential for medical applications, for example in interventional angiology and cardiology.

The seminar will be held online on Thursday, October 19, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

15. 6. 2023 Luka Gnidovec (MSc student at FCCT UL): Structural aspects of hnRNP H binding to G₄C₂ hexanucleotide repeats

Abstract

ALS is a fatal neurodegenerative disease characterized by gradual degeneration and eventual loss of motor neurons, which results in progressive muscular paralysis. Even though there are multiple known genetic mutations that contribute to the disease, the molecular mechanisms of ALS are still largely not understood. One of the most common causal mutations in ALS is a hexanucleotide repeat expansion (HRE) GGGGCC in the gene C9ORF72. Due to its high G content, this sequence repeats can form higher order structures, including multiple types of G-quadruplexes (GQs). There are many pathways through which HRE in C9ORF72 could initiate neurodegeneration. Aberrant multivalent interactions of RNA with RNA binding proteins (RBPs) could cause sequestration of RBPs in nuclear and cytoplasmic foci, inhibiting their physiological function. Splicing regulator hnRNP H was identified as one of the main interactors of G₄C₂ nucleotide repeats. The nature of hnRNP H binding to G₄C₂ repeats remains ambiguous. In my master’s thesis, we wanted to determine the structural aspects of hnRNP H binding to G₄C₂ repeats. First, we aimed to show a preference in binding to either folded or unfolded GQs. Next, we were interested if hnRNP H possesses the ability to linearize folded GQs. Using thermophoresis and fluorescence quenching, we showed that there seems to be a preference for hnRNP H binding to unfolded G₄C₂ motifs and that hnRNP H does not linearize GQs in the tested concentration range. EMSA assay results also implicate formation of larger structures. We speculate this is caused by condensation of hnRNP H and G₄C₂ repeats, which could occur through liquid-liquid phase separation.

The seminar will be held online on Thursday, June 15, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

15. 6. 2023 Klementina Polanec (MSc student at FCCT UL): Analysis of biotin identification targets of ORF1p and optimization of LINE1 retrotransposition assay with vtRNA and YRNA

Abstract

Retrotransposon LINE1 represents up to 17% of the human genome. It is the only autonomous mobile genetic element still active in humans. Full-length LINE1 element includes 3 open reading frames for ORF1p, ORF2p, and ORF0p. Increased levels of retrotransposon activity have been associated with several neurodegenerative diseases and cancer. A comprehensive understanding of LINE1 retrotransposition is essential for the development of potential therapeutics. Therefore, it is important to discover proteins and RNA molecules that interact with LINE1 components (especially ORF1p) and modulate retrotransposition. In our work we investigated the effect of RNY and vtRNA on LINE1 retrotransposition and analysed the mass spectrometry results of biotin identification of potential ORF1p interaction partners. Using retrotransposition assay we showed that ncRNA RNY and vtRNA have a statistically significant inhibitory effect on LINE1 retrotransposition, reducing it by 25%. We observed the same inhibitory effect in HeLa and HEK293T cell lines. Mass spectrometry results of biotin identification of ORF1p revealed that potential ORF1p interaction partners, like ORF1p, localize in stress granules, cytoplasm, nucleus and nucleoli. Potential interactors are mainly RNA-binding proteins involved in various steps of RNA metabolism. By immunodetection, we confirmed 5 ORF1p interactors among which TDP-43 and FUS are strongly implicated in different neurodegenerative diseases. IGF2BP1 and ELAVL1 also colocalized with transiently expressed Flag-ORF1p in unstressed HEK293T cells. Our results contribute to a better understanding of LINE1 and the factors regulating retrotransposition.

18. 5. 2023 Veno Kononenko (PhD, researcher at the Biotechnical Faculty): Gelatin nanoparticles-loaded with analog of marine toxin 3-alkylpyridinium salt APS7: a promising support in human lung cancer therapy

Abstract

In our research, we addressed the problem of chemotherapy resistance and off-target effect in the treatment of lung cancer. Activation of nicotinic acetylcholine receptors (nAChRs), the ligand-gated ion channels, is associated with increased lung cancer cell proliferation and decreased chemotherapy-induced apoptosis, leading to prevention of successful cancer treatment. We hypothesize that inhibition of nAChR by nicotinic antagonists may have beneficial effects on lung cancer treatment. In our study, the effects of 3-alkylpyridinium salt APS7, a synthetic analog of natural 3-alkylpyridinium polymers isolated from the marine sponge Haliclona (Rhizoneira) sarai, were tested on A549 human lung adenocarcinoma cells. To ensure that APS7 is preferentially taken up by cancer cells, a nanodelivery system, gelatin nanoparticles loaded with APS7 (APS7-GNPs), was synthesized by nanoprecipitation method. Our results show that free APS7 causes similar concentration-dependent cytotoxicity for A549 lung cancer cells and for the non-tumorigenic lung epithelial cell line BEAS-2B, whereas APS7-GNPs are cytotoxic only for A549 cancer cells. Both free APS7 and APS7-GNPs (at subcitotoxic concentrations) inhibited nicotine-induced Ca2+ influx in A549 cells, suggesting that APS7 acts as an antagonist of nicotine and that gelatin nanoparticles do not prevent the antagonistic effect of APS7. We demonstrated that nicotine can reduce the cisplatin-induced apoptosis and increase the reproductive capacity of cisplatin-treated A549 cells and that these effects of nicotine can be attenuated by APS7 and APS7-GNPs. Compared with free APS7, the APS7-GNPs showed a stronger effect in reducing reproductive capacity of A549 cells in a 2-week experiment, suggesting that APS7-GNPs exert a longer-lasting effect on the cancer cells than free APS7. Our results indicate the potential of APS7 and APS7-GNPs for use in combination with existing chemotherapeutic agents in the treatment of lung cancer.

The seminar will be held online on Thursday, May 18, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

20. 4. 2023 Špela Koren (PhD student at JSI): Lipid trafficking and oxidation under ferroptic conditions: A lipidomic approach

Abstract

Lipid droplets (LDs) are cytosolic organelles that manage the uptake, storage, and utilization of fatty acids in the cell. The objective of this study is to investigate the role of LDs in regulating ferroptosis, a form of programmed cell death triggered by the oxidative damage of polyunsaturated fatty acid (PUFA)-containing phospholipids in cell membranes. Given the central role of LDs in fatty acid metabolism and storage, it is plausible that LDs also play a role in regulating the generation and turnover of oxidized lipids during ferroptosis. To investigate this hypothesis, we have established a collaboration with Dr. Maria Fedorova's laboratory in Dresden, which specializes in lipidomics, epilipidomics, and bioinformatics, and has vast expertise in studying lipid metabolism and oxidative stress in cellular systems. Our study aims to combine their expertise with our own to obtain a lipidome-scale view of lipid trafficking and oxidation in MDA-MB-231 breast cancer cells under controlled ferroptotic conditions. We will use lipidomic and epilipidomic approaches to identify and quantify changes in the lipidome and its dynamics in cells with impaired LD turnover. Additionally, we will also employ bioinformatic tools to integrate and analyze the complex lipid data generated in the study. The findings of our study have the potential to reveal novel mechanisms underlying LD-mediated regulation of ferroptosis and provide insights into the complex interplay between lipid metabolism and ferroptotic cell death.

The seminar will be held online on Thursday, April 20, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

20. 4. 2023 Larisa Lara Popošek (PhD student at BF): The quest for new lipid-binding proteins and biotechnological applications

Abstract

Aegerolysins are relatively small proteins (15 kDa) with unique biotechnological potential (1,2). The main biochemical characteristic of aegerolysins is their high affinity for specific membrane lipids or lipid mixtures (1-3). In addition, they are non-toxic, and protocols for their isolation in recombinant form have already been developed (4,5). They can be found mainly in the kingdom of fungi and bacteria (3). In some production organisms, aegerolysins co-occur with MACPF (Membrane Attack Complex / Perforin) protein partners. Aegerolysins in combination with protein-protein form pores in target lipid membranes (3,4). The best studied aegerolysins and their protein partner are those from the fungal genus Pleurotus (4-10).

So far known aegerolysins are characterized by high affinity for the lipid receptor ceramide phosphoethanolamine (CPE), which is characteristic for some insects (1). In addition, some of these aegerolysins less specifically bind to the mixture of sphingomyelin (SM) and cholesterol (Hol), which is characteristic lipid mixture in vertebrate membranes (4). Fluorescently labeled aegerolysins can be used to study these lipids, in the similar manner as antibodies are used to study the localization or presence of proteins in the cells (5, 11). Although aegerolysins are non-toxic, they may, in the presence of a protein with the MACPF domain, become part of a cytolytic complex that perforates the membrane of those cells that contain the lipid receptor recognized by aegerolysin (1,4,5,12). Aegerolysins are therefore biocomponent cytolysins - component A (aegerolysin) recognizes the lipid target, and component B (protein partner with the MACPF domain) further allows the formation of pores in the target membrane.

We are currently studding 8 novel aegerolysins and their MACPF protein partners. Among them 4 aegerolysins, and their native MACPF-containing protein partner are coded by P. pulmonarius. The other four aegerolysins and their MACPF protein partners are coded by basidoimicetes Heterobasidion irregulare, Trametes versicolor, Lepista nuda and Mucidula mucida. We have tried obtain all these proteins in recombinant and the first experiments have been performed. You will therefore find out in the seminar: (i) whether these novel aegerolysins also recognize lipid receptors, (ii) whether do these aegerolysins in combination with MACPF protein partners show any hemolytic activity and whether they are (iii) insecticidal.

Panevska, A. et al. Aegerolysins from the fungal genus Pleurotus – Bioinsecticidal proteins with multiple potential applications. J. Invertebr. Pathol. 2020.

Gundner, M. et al. What can mushroom proteins teach us about lipid rafts. Membranes 2021.

Butala, M. et al. Aegerolysins, lipid-binding proteins with versatile functions. Semin. Cell Dev. Biol. 2017.

Ota, K. et al. Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-
formation by a MACPF/CDC-likepore-forming protein, pleurotolysin B. Biochimie 2013.

Skočaj, M. et al. Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein. PLoS ONE 2014.

Berne, S. et al. Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal
fruiting. Biochim Biophys Acta BBA Gen. Subj. 2002.

Sepčić, K. et al. Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains. FEBS Lett. 2004.

Tomita, T. et al. Pleurotolysin, a novel sphingomyelinspecific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex. J. Biol. Chem. 2004.

Bhat, H.B et al. Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and
cholesterol-rich membrane domains. J. Lipid Res. 2013.

Shibata, T. et al. Isolation and characterization of a novel two-component hemolysin, erylysin A and B, from an edible mushroom, Pleurotus eryngii. Toxicon. 2010.

Bhat, H.B., et al. Evaluation of aegerolysins as novel tools to detect and visualize ceramide
phosphoethanolamine, a major sphingolipid in invertebrates. FASEB J. 2015.

Resnik, N. et al. Highly selective anti-cancer activity of cholesterol-interacting agents methyl-β-cyclodextrin and ostreolysin A/pleurotolysin B protein complex on urothelial cancer cells. PLoS ONE 2015.

16. 3. 2023 Alenka Vesel (MSc student at BF): Interaction of recombinant proteins Cry34Ab1 and Cry35Ab1 from bacteria Bacillus thuringiensis with artificial lipid membranes

Abstract

Colorado beetle (Leptinotarsa decemlineata) and corn rootworm (Diabrotica sp.) are important corn pests that cause annual losses of over a billion dollars in the USA. Beetle populations are mostly reduced with use of chemicals pesticides, which have a plethora of negative effects on people, other non-target organisms and the environment. A more recent approach to managing beetle populations is implementing the use of transgenic species of potato and corn hybrids, which carry genes for crystal insecticidal proteins (Cry) synthesis from Bacillus thuringiensis. Mechanism of action of Cry toxins consists of them binding to specific protein receptors in insect midgut epithelium, followed by pore formation, cell lysis which results in insect dying. Binary complex Cry34Ab1/Cry35Ab1 is one option for reducing corn rootworm larvae population. Cry34Ab1 has features of aegerolysin family, which are small (15-20 kDa), β-structural proteins and can be found in different bacterial and fungal genera. Proteins from aegerolysin family can recognize and bind to different lipids or combination of lipids. It was shown that complex Cry34Ab1/Cry35Ab1 can permeabilize artificial phospholipid membranes, which indicates a possibility of lipid receptor. To get a deeper insight into mechanism of action of Cry34Ab1/Cry35Ab1 we studied interaction of said complex with artificial lipid membranes, prepared from commercial or natural lipids. We successfully expressed and purified Cry34Ab1 and Cry35Ab1 from competent E. coli cells. On surface plasmon resonance we saw response when adding Cry34Ab1/Cry35Ab1 to PG/POPC/Chol large unilamellar vesicles (LUV), but we saw two-fold increase in response when adding the complex to PG/PE/Chol lipid membrane. To test the permeabilization we performed calcein release test on the same vesicle combination and release of calcein was only detected in PG/PE/Chol vesicles, when adding the Cry complex. Cry34Ab1/Cry35Ab1 also reacts with Sf9 insect total lipid extract and causes permeabilization in calcein release test. After cryo-electronic microscopy we can see that Cry34Ab1 and Cry35Ab1 do form pore-like structures on PG/PE/Chol LUV.

The seminar will be held online on Thursday, March 16, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

16. 3. 2023 Adrijan Ivanušec (PhD student at JSI): Group IIA secreted phospholipase A₂ and Alzheimer’s disease?

Abstract

Alzheimer’s disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria. Similar effects can be also observed in the motor neurons after intoxication with an orthologous GIIA from snake venom, β-neurotoxic ammodytoxin (Atx). To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant Atx and rat GIIA (rGIIA) and their enzymatically inactive mutants, Atx(D49S) and rGIIA(D49S). We demonstrated that all four of them interact with the subunit II of cytochrome c oxidase (CCOX-II). rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Both Atx(D49S) and rGIIA(D49S) also exerted an inhibitory effect on mitochondrial membrane potential and CCOX activity in PC12 cells and rat brain tissue sections, respectively. To study the relevance of the enzymatic activity of Atx in cell internalisation and subsequent intracellular movement, we labelled Atx(D49S) with gold nanoparticles (GNP), and incubated it with PC12 cells. Using transmission electron microscopy, we observed that Atx(D49S)-GNP internalised into the cells. There, it was detected in different vesicle-like structures, cytosol, endoplasmic reticulum and mitochondria, where it was spotted in the intermembrane space and matrix. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity, as determined with confocal microscopy. Our results show that the effects of mammalian and snake venom β-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.

16. 2. 2023 Tadeja Bele (PhD student at JSI and BF): Antagonists of α7-nicotinic acetylcholine receptor and their potential therapeutic effects on lung cancer cells

Abstract

Receptors and their signalling cascades play a crucial role in regulating cellular processes, and their dysregulation leads often to disease development. In particular, elevated expression of nicotinic acetylcholine receptors (nAChRs) has been observed in various types of cancer, including lung cancer. The binding of ACh or other agonists, such as nicotine and his derivatives, to these receptors has been linked to uncontrolled cell division, prevention of apoptosis and induction of angiogenesis, ultimately supporting tumour growth and metastasis. In contrast, antagonists of nAChRs have shown opposite effects on cells, suggesting their potential value in cancer therapy. Vulfius et al. (2014) showed that secreted phospholipases A₂ (sPLA₂s) from Elapidae and Viperidae snake venoms act as antagonists of nAChRs by supressing ACh-elicited ion currents. Such effect was independent of phospholipase activity. Several human orthologues of snake venom sPLA₂s exist and we were interested whether or not their physiological function is also regulation of nAChR activity. We prepared recombinant human sPLA₂s, group V (hGV) and X (hGX), as well as their single point active site mutants devoid of enzymatic activity. Electrophysiological assessment of their effects on α7 and adult muscle type nAChRs expressed in Xenopus laevis oocytes were performed. All four human sPLA₂ molecules inhibited ion current that was triggered by ACh through α7 nAChR. Interestingly, the analysis revealed absolute selectivity of enzymatically inactive hGV for α7 nAChRs. We are trying to identify specific nAChR-related signalling pathways affected by these proteins. As well, the preliminary results of testing the effects of human sPLA₂ molecules on proliferation and death of various lung cancer cell lines and BEAS-2B, a human non-tumorigenic lung epithelial cell line, will be presented.

The seminar will be held online on Thursday, February 16, starting at 13:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

16. 2. 2023 Kity Požek (PhD student at JSI): Advancement on the study of thrombocytopenia-inducing Vaa-snaclec-3/2 from the venom of the nose-horned viper

Abstract

Thromboembolic complications during surgical treatment of myocardial infarction and ischemic stroke are a pressing challenge in interventional angiology and cardiology. Platelets play a pivotal role in the response to vascular injury during surgery. They adhere to injured vessels, become activated, aggregate, secrete mediators that promote platelet activation, and promote blood clotting. Antithrombotics currently used to prevent thromboembolic complications, on the other hand, increase the likelihood of bleeding and impair wound healing after surgery, so they are not ideal and it is necessary to develop antiplatelet drugs without such adverse effect. Venom of the nose-horned viper (Vipera a. ammodytes) was observed to induce thrombocytopenia, a profound decrease in the platelet count. However, we noticed that upon administration of the antivenom, the platelet counts rapidly increased again and, most importantly, platelets remained functional, so there was no risk of bleeding. Our analysis showed that thrombocytopenia was induced by a fraction of the venom containing non-enzymatic dimeric C-type lectin-like proteins (snaclecs). We purified a number of snaclecs from the crude Vaa venom and tested them for their effects on platelet agglutination and aggregation, platelet activation, and binding to platelet receptors. In vitro studies revealed that Vaa-snaclec-3/2 was most likely to induce a thrombocytopenic effect as it attenuated platelet agglutination through its interaction with the platelet GPIb receptor. In collaboration with our colleagues from the Veterinary Faculty, University of Ljubljana, we are currently testing the effects of this protein in an in vivo study using a mouse model of arterial thrombosis. Our aim is to evaluate its ability to prevent blood clot formation and arterial occlusion after experimentally induced vascular injury, which is the first step towards its potential medical application. The preliminary results we will present are very promising.

19. 1. 2023 Neža Repar (PhD, researcher at the Biotechnical Faculty): Developing a model to test microplastic impact on lung epithelial barriers formation and functionality

Abstract

Nano- and microplastics (NMP) are plastic particles smaller than 5 mm. They can be manufactured for a specific purpose or released through the degradation of plastics and NP /MP-containing products. NMPs are widely distributed in the environment. The two major routes of exposure for humans are ingestion and inhalation of indoor air. Studies have already reported that airborne particles can deposit or accumulate in the lungs and cause occupational diseases such as lung cancer in textile workers.
The objective of this study is to evaluate the effects of NMP on human health with particular emphasis on the integrity and functionality of the lung barrier. Since a good foundation for a project is a solid model, we attempted to mimic lung tissue by growing A549 cells on PET inserts under either submersion or air-liquid interface (ALI) conditions, the latter being more similar to the natural environment in the alveoli.
We then characterized our model in detail using various methods prior to MP exposure. In particular, we observed in time-course experiments that transepithelial electrical resistance (TEER) and epithelial permeability (estimated by the Lucifer Yellow assay) increased and decreased, respectively, with time and reached a plateau 10 days after seeding, indicating the formation of an epithelial barrier. In further experiments, we instead examined and compared surfactant production and tight junction expression in A549 cells grown under either submerged or ALI conditions. This showed that ALI produced more surfactant (drop spreading method) and occludin (confocal microscopy) compared with submerged cells, with no significant difference in TEER and permeability values, suggesting that ALI-grown cells provide a more realistic model for lung exposure. A549 cells were then exposed to NMPs, and our results showed no clear effects of NMPs on cell viability.

The seminar will be held online on Thursday, January 19, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

19. 1. 2023 Maja Hostnik (PhD student at the Biotechnical Faculty): Phage GIL01 influence on Bacillus thuringiensis sporulation

Abstract

GIL01 infects endospore-forming bacterium B. thuringiensis serovar israelensis that has been used as an efficient biopesticide worldwide. Following infection, GIL01 prophage enters lysogenic cycle and resides within the host as autonomous linear plasmid without integrating into the chromosome. Lysogenic phages generally establish a stable relationship with their host bacteria and can in some cases confer novel properties, such as virulence traits or increased survival/ecological fitness, to the bacterial lysogens. Since GIL01 genome is a small extracellular replicon, we assumed its genome could be incorporated inside of a bacterial endospore. Bacterial endospores can serve as phage genome protection shells against various environmental factors and can therefore enhance their survival in unfavourable environments.
The aim of our work was to evaluate potential influence of GIL01 on sporulation of its host B. thuringiensis serovar israelensis. For this purpose, a B. thuringiensis serovar israelensis plasmid cured (nonlysogenic) strain and an entomopathogenic B. thuringiensis serovar israelensis strain isolated from soil in Brazil were used. Sporulation rates of strains harbouring or not the GIL01 prophage were evaluated. The results show that GIL01 lysogeny has a significant influence on the sporulation rate in plasmid-cured B. thuringiensis strain, while the phage influence in the entomopathogenic strain is minor. We also confirmed the majority of spores carry GIL01 genome. Currently, we are investigating mechanisms underlying GIL01 influence on B. thuringiensis sporulation.

15.12. 2022 Kity Požek (PhD student at JSI): Serine protease VaaSP-6 from the nose-horned viper venom and its effect on blood coagulation

Abstract

The pathological effect of the nose-horned viper (Vipera a. ammodytes; Vaa) venom is mainly directed towards the blood coagulation system or hemostasis. Among protein families represented in the Vaa venom, serine proteases are the most markedly hemotoxic. They usually act as anticoagulants, less commonly, as in the case of VaaSP-VX, also as procoagulants (Latinović et al., 2020). VaaSP-VX is the first known enzyme that accelerates blood coagulation by activating both coagulation factor X (FX) and FV. Such unique activity makes this molecule promising for diagnostic and therapeutic applications. We sequenced VaaSP-VX only partially (40%). Using this sequence information, we were unable to trace its cDNA in the cDNA library of the Vipera a. ammodytes venom gland. We however spotted and sequenced a cDNA of a very similar venom serine protease, VaaSP-6, likely the isoform of VaaSP-VX. Due to the high sequence similarity, we speculated that both molecules might have also similar pharmacological activities. To test this, we isolated VaaSP-6 from the Vaa venom in three chromatographic steps – gel filtration, cation-exchange, and reversed-phase high-performance liquid chromatography. We determined its basic biochemical properties and studied its effects on blood coagulation by performing standard clinical coagulation assays. Unexpectedly, this 35-kDa glycosylated protein displayed anticoagulant and not procoagulant activity as VaaSP-VX. Consistently, VaaSP-6 was unable to activate FX in vitro. We are in the process of deciphering the details of its mechanism of action and identifying the key structural differences between VaaSP-6 and VaaSP-VX that determine whether the serine protease will act anticoagulantly or procoagulantly.

Latinović, Z., Leonardi, A., Koh, C. Y., Kini, R. M., Bakija, A. T., Pungerčar, J., & Križaj, I. (2020). The procoagulant snake venom serine protease potentially having a dual, blood coagulation factor V and X-activating activity. Toxins, 12(6), 1–16.

The seminar will be held online on Thursday, December 15, starting at 13:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

17. 11. 2022 Katja Doberšek (MSc student at JSI); Tina Zavodnik (MSc student at JSI); Gašper Žun (PhD student at JSI); Mauro Danielli (Postdoc. Assoc. at JSI): SARS-CoV-2 ORF8 protein's human protein interactors and covid-19 pathology

Abstract

SARS-CoV-2 is the virus that has infected the highest number of people in the shortest time in the past few years. The ssRNA genome of the virus encodes 29 proteins - four structural proteins (spike, envelope, membrane and nucleocapsid), sixteen non-structural proteins and nine accessory proteins. ORF8 is one of the accessory proteins that has been suggested to interfere with the hosts immune response. Deletion of ORF8 was shown to be correlated with milder disease, lower incidence of hypoxia and better disease outcome. In our study we tested binary protein-protein interactions of ORF8 with predicted human interactors from previous large-scale studies, and examined the ability of various ORF8 variants to bind to these proteins, using the yeast-two hybrid method. We then analyzed the effect of infection with SARS-CoV-2 strains with those ORF8 variants on the pulmonary function of the affected patients. We also analyzed the effect of target proteins knock-down in cell culture models of SARS-CoV-2 infection and pathogenesis.

The seminar will be held online on Thursday, November 17, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

20. 10. 2022 Anja Pavlin (PhD student at BF): Gp1: A novel repressor of the temperate phage GIL01 lytic cycle

Abstract

GIL01 is a temperate phage that infects the insect pathogen Bacillus thuringiensis. As a temperate phage, it can choose between two life cycles after infection of its host: a dormant lysogenic cycle, in which transcription of phage genes is repressed, or an active lytic cycle, in which new virions are formed and released from the cell by host lysis. The regulation of these two distinct life cycles is not well studied, with the exception of the model temperate phage λ. Transcription of phage GIL01 genes is controlled by three promoters. The weak and strong tandem promoters, P1 and P2, respectively, control transcription of gene cluster required for replication of the phage genome and regulation of phage life cycles, while P3 controls expression of phage structural genes and genes required for bacterial lysis. While most temperate phages encode their own repressor that silences gene expression during the lysogenic cycle, GIL01 hijacks bacterial protein LexA, repressor of the SOS response that bacteria use to respond to DNA damage. In our previous studies, we identified gp7, a small phage protein that forms a complex with LexA and enhances its binding to operators at the P1 and P3 promoters and is essential for maintaining the lysogenic cycle. In addition to gp7, gp1, another small protein, has also been identified as critical for establishing and maintaining the lysogenic state, but with an unknown mechanism of action. In this work, we show that gp1 binds to the operator at the P2 promoter of GIL01 and forms a filament that prevents binding of the RNA polymerase and thus transcription from P2. P2 is the stronger promoter of the P1-P2 tandem controlling genes required for replication and regulation of the phage genome. Gene for gp1 is the first phage gene to be transcribed after infection, creating a negative feedback loop. While most temperate phages insert into the host genome and replicate along with the bacterial chromosome, GIL01 resides in the host cell as an extrachromosomal linear plasmid in 10-15 copies per cell. We therefore believe that gp1 is the safety net of phage GIL01, maintaining sufficient copy number to ensure its transmission to the daughter cell.

The seminar will be held online on Thursday, October 20, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

20. 10. 2022 Leja Perne (MSc student at JSI): Lipid droplets and the regulation of ferroptosis in cancer cells

Abstract

Ferroptosis is a recently discovered form of regulated cell death, caused by excessive lipid peroxidation – a harmful process that affects (poly)unsaturated membrane lipids and ultimately destroys cellular membranes. Ferroptosis can be induced in cells by impairing the function of anti-ferroptotic enzymatic systems that catalyse the reduction of oxidized lipids and halt lipid peroxidation. This could be a potential strategy for anti-cancer therapy as many cancers show sensitivity to ferroptosis induction. Lipid droplets (LDs), cytosolic fat-storage organelles, display emerging roles in the regulation of (poly)unsaturated fatty acid (FA) trafficking and the maintenance of lipid membrane saturation. As the distribution and accessibility of (poly)unsaturated FAs in the cells and the degree of saturation of lipid membranes significantly influences the sensitivity of cells to ferroptosis induction, this suggests a possible connection between LDs and ferroptosis, which is not yet understood. Here, we investigated the role of LDs in MDA-MB-231 breast cancer cells under ferroptotic conditions by inhibiting LD biogenesis with pharmacological inhibitors of diacylglycerol acyltransferase (DGAT). Using confocal microscopy and flow cytometry, we found that LDs are important regulators of lipid peroxidation and ferroptosis sensitivity in MDA-MB-231 breast cancer cells. The ability of LDs to regulate ferroptosis sensitivity suggests that targeting of LD turnover and the anti-ferroptotic systems shows great potential to be a valid therapeutic strategy.

22. 9. 2022 Gašper Žun (PhD student at JSI): Highlights of CRISPR-Cas9 in yeast Saccharomyces cerevisiae

Abstract

Recent developments of CRISPR have proven the tool to be one of the most promising techniques for marker-free genome modifications. Moreover, the budding yeast S. cerevisiae, among others, possesses exceptionally effective DNA-repair pathway via homologous recombination and is therefore particularly suitable for such genetic studies. Targeting one locus at a time with CRISPR-Cas9 system, following by repair via homologous recombination using offered template DNA, usually results in up to 100% modified genomes. We developed a technique to induce several double-strand breaks within different loci simultaneously, i.e. multiplex CRISPR-Cas9. Providing templates for homologous recombination, the technique exhibits adequate efficacy for marker-free genome modifications. Furthermore, off-target effects of CRISPR-Cas9 system were quantified within its hotspot (around PAM-sequence) and throughout the whole genome. Finally, a case study, published in Nature Ecology and Evolution, is presented. CRISPR-Cas9 method was applied for successful marker-free allele swap of quantitative trait nucleotides. Candidate QTNs of sporulation efficiency were proposed by genome-wide association studies and further quantified by allele swap within isogenic backgrounds. CRISPR-Cas9 is a powerful tool and applying it on multiplex level possesses even greater ability to construct variants with advanced phenotypes.

The seminar will be held online on Thursday, September 22, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Jože Pungerčar (joze.pungercar@ijs.si) to receive a link.

16. 6. 2022 Klavdija Fortuna (PhD student at BF): Bioinsecticidal potential of aegerolysin proteins from mushrooms of the genus Pleurotus and their mutants

Binding of Pleurotus aegerolysins to invertebrate-specific sphingolipid ceramide phosphoethanolamine (CPE) and the formation of transmembrane pores in association with pleurotolysin B (PlyB) suggest the possibility of using aegerolysin/PlyB complexes as novel biopesticides as well as producing genetically modified crops resistant to from an economical point of view important pests. Erylysin A (EryA) is the only known fungal aegerolysin that specifically interacts with CPE-enriched membranes. By isolating the single point mutant, EryA E69A, we sought to eliminate cholesterol-specific binding and increase the affinity for lipid vesicles. The mutant could not be obtained so its further characterization was not possible. According to previous research, the OlyA6/PlyB complex has the strongest lytic activity of all aegerolysin/PlyB complexes studied and an even stronger insecticidal activity could be shown by using the OlyA6 E69A mutant. Both versions of the protein do not exhibit lytic activity in the absence of the PlyB protein partner, while in combination with PlyB they are both lytic even at low concentrations when cholesterol (Chol) is present in the membranes. The binding affinity of the mutant to Chol-free vesicles is increased compared to the wild type protein, while the OlyA6 E69A/PlyB complex showed no greater permeabilization of biological membranes compared to the OlyA6/PlyB complex. We confirmed that OlyA6 E69A recognizes different CPE conformations in biological membranes, confirming that this mutant could be biotechnologically very important as many insects are auxotrophic for Chol.

The seminar will be held on-line on Thursday June 16, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

16. 6. 2022 Ana Maklin (Master student at FCCT): The influence of Sig-1R on aggregation of TDP-43 in neurodegeneration

Neurodegenerative diseases affect millions of people worldwide. The symptoms of neurodegeneration are caused by the progressive loss of neurons in the central or peripheral nervous system. Protein aggregation into insoluble inclusion bodies is one of the main hallmarks of neurodegeneration. TAR (TransActivation Response) DNA-binding protein 43 (TDP-43) is one of the critical proteins in neurodegeneration. In normal cells, TDP43 is mainly present in the nucleus and plays an important role in RNA regulation. Under pathological conditions, cleavage, hyperphosphorylation and ubiquitination of TDP-43 can occur. These posttranslational modifications lead to cytoplasmic accumulation and aggregation of TDP-43. Another critical protein in neurodegeneration is the chaperone at the endoplasmic reticulum (ER) Sigma receptor 1 (Sig-1R). Sig-1R and TDP-43 are interdependent in the regulation of cellular protein quality control pathways. Dysregulation of TDP-43 can be directly caused by Sig-1R aggregation. ER chaperone Sig-1R accumulates in surviving motor neurons in ALS and may have neuroprotective functions. We isolated soluble TDP-43 as a fusion protein with MBP (maltose-binding protein) and Sig-1R protein lacking N-terminal transmembrane region (ΔNSig-1R) for further aggregation experiments. To analyze TDP-43 aggregation, we utilized spectrofotometric turbidity measurements (optical density at 395 nm). Measurements were initiated by cleavage of MBP from TDP-43_MBP with the protease TEV. Aggregation was followed in the presence of a previously known disaggregase (tRNA) and a hypothetical disaggregase (ΔNSig-1R). Addition of tRNA decreased or prevented aggregation of TDP-43, and the rate of aggregation depended on the amount of tRNA added. Addition of 4 and 8 μM tRNA prevented aggregation, 2 μM tRNA delayed and decreased aggregation, 1 and 0,5 μM tRNA also decreased aggregation of TDP-43 compared to TDP-43 without tRNA. Addition of 4 and 8 μM ΔNSig-1R did not prevent TDP-43 aggregation, the plateau of aggregation curve was higher than without ΔNSig-1R. By measuring the turbidity, we could follow the aggregation of TDP-43 and the role of different disaggregases in aggregation. We were not able to show that ΔNSig-1R had any effect on TDP-43 aggregation.

16. 6. 2022 Jerneja Nimac (Master student at FCCT): Protein interactions of human MATR3

Matrin 3 (MATR3) is a DNA/RNA-binding nuclear matrix protein involved in several cellular processes. These include early stages of DNA damage response, transcription, mRNA stability, alternative splicing, and mRNA export. Mutation S85C in the MATR3 gene is associated with the development of a slowly progressive form of amyotrophic lateral sclerosis (ALS). However, the role of MATR3 in the pathogenesis of this disease is unknown. Moreover, little is known about the protein-protein interactions of MATR3 and its mutants. Therefore, we aimed to identify proteins that interact with human MATR3 and its mutant MATR3S85C in vivo using the BioID method, which is based on labelling proteins in proximity with biotin. To this end, we generated cell lines stably expressing the fusion protein of MATR3 or MATR3S85C with the biotin ligase BioID2 or BioID2 without a fusion partner. We found that the fusion proteins of MATR3 and MATR3S85C with the biotin ligase BioID2 have the same cellular localization as endogenous MATR3 and that BioID2 is enzymatically active. After examining the BioID2 activity and cellular localization of the expressed proteins, we successfully isolated biotinylated interaction partners of MATR3 and MATR3S85C from cell lysates by the pull-down assay. Using silver staining and immunodetection of the biotinylated proteins after Western blotting, we detected proteins specific for samples with MATR3 and MATR3S85C but not for samples with BioID2 without a fusion partner. We then identified these interaction partners by liquid chromatography – mass spectrometry. Among the identified proteins are already known interactors of MATR3 and its mutant S85C as well as new, yet unknown ones that could contribute significantly to the understanding of ALS.

19. 5. 2022 Mia Žganjar (PhD student at BF and JSI): Wild yeast selection and identification for microbial oils production from short-chain fatty acids (SCFAs)

Abstract

Commercial production of microbial oils has not yet reached an economically feasible turning point for a progressive fossil fuels replacement. An economically viable process could include the oils derived from storage lipids, synthetized in oleaginous yeasts, and produced from organic wastes via bacterial anaerobic fermentation step to short-chain fatty acids (SCFAs) and an additional yeast fermentation step to microbial oils.
For this purpose, over 1400 wild yeast strains were selected for screening of effective growth on SCFAs as a carbon and energy source. The screening was conducted on solid media compositions, consisting of three SCFAs: acetic acid (C2), propionic acid (C3) and butyric acid (C4).
Growth parameters were computed with the Python pyphe package (v0.981) and compared to the control oleaginous strain CECT1240; Yarrowia lipolytica. Yeast capable of growing on SCFAs were further tested for their ability to accumulate neutral lipids to high quantities. For this, a semi-high-throughput approach, based on the Nile red dye staining was used.
Among 1438 candidate wild yeast strains, 11 were exceeding the control strain in both required traits – effective growth on SCFAs and high lipid accumulation. In addition to the already known oleaginous potential of yeast from the genus Yarrowia, we found several new and promising yeast strains capable of the described biomass feedstock conversion in the genus Pichia. Therefore, we show an effective approach to obtain new biotechnologically and economically promising strains for the competitive microbial oil production.

The seminar will be held on-line on Thursday May 19, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

19.5.2022: Klavdija Fortuna (PhD student at BF): Bioinsecticidal potentialof aegerolysin proteins from mushrooms of the genus pleurotus and their mutants

Binding of Pleurotus aegerolysins to invertebrate-specific sphingolipid ceramide phosphoethanolamine (CPE) and the formation of transmembrane pores in association with pleurotolysin B (PlyB) suggest the possibility of using aegerolysin/PlyB complexes as novel biopesticides as well as producing genetically modified crops resistant to from an economical point of view important pests. Erylysin A (EryA) is the only known fungal aegerolysin that specifically interacts with CPE-enriched membranes. By isolating the single point mutant, EryA E69A, we sought to eliminate cholesterol-specific binding and increase the affinity for lipid vesicles. The mutant could not be obtained so its further characterization was not possible. According to previous research, the OlyA6/PlyB complex has the strongest lytic activity of all aegerolysin/PlyB complexes studied and an even stronger insecticidal acti vity could be shown by using the OlyA6 E69A mutant. Both versions of the protein do not exhibit lytic activity in the absence of the PlyB protein partner, while in combination with PlyB they are both lytic even at low concentrations when cholesterol (Chol) is present in the membranes. The binding affinity of the mutant to Chol-free vesicles is increased compared to the wild type protein, while the OlyA6 E69A/PlyB complex showed no greater permeabilization of biological membranes compared to the OlyA6/PlyB complex. We confirmed that OlyA6 E69A recognizes different CPE conformations in biological membranes, confirming that this mutant could be biotechnologically very important as many insects are auxotrophic for Chol.

21. 4. 2022 Adrijan Ivanušec (PhD student at JSI): Interaction between secreted phospholipases A2 and mitochondria

Abstract

Secreted phospholipase A2 group IIA (GIIA sPLA2) plays both physiological and pathological role in mammalian brain. Physiologically, it is involved in the regulation of neurotransmission, neuritogenesis and mitochondrial homeostasis, while pathologically; it is implicated in neurodegenerative and cerebrovascular diseases. However, molecular mechanisms of action of GIIA sPLA2 in these effects are not clear but the interaction of GIIA with neuronal mitochondria seems to play an important role. To get a deeper insight into the interaction of GIIA sPLA2 with mitochondria, we synthesized a recombinant rat GIIA sPLA2 (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S) and assessed their actions on neuronal mitochondria. Heterologous competition assay using radioiodinated Atx showed that rGIIA binds to the subunit II of cytochrome c oxidase (CCOX-II), an essential constituent of the respiratory chain complex, which was previously identified as the mitochondrial receptor of ammodytoxin (Atx), a β-neurotoxic orthologue of rGIIA from the snake venom. The affinity of rGIIA for CCOX-II was in the μM range, about 100-fold lower than that of Atx. Nevertheless, rGIIA and rGIIA(D49S) potently inhibited CCOX activity in isolated rat mitochondria, indicating that they interact with CCOX in a slightly different way than Atx. rGIIA(D49S) inhibited mitochondrial CCOX activity also in intact PC12 cells and in rat brain tissue sections as demonstrated by flow cytometry and histochemical staining, respectively. Previously, it has been established that rGIIA is an enzyme resident to mitochondria, but as we demonstrated using fluorescently-labelled rGIIA and confocal laser microscopy, it is also able to reach mitochondria in PC12 cells from extracellular space similarly to exogenous, snake venom sPLA2. The internalization into the cells seems to be independent of the sPLA2 enzymatic activity, since rGIIA(D49S) was also able to enter the cells. Altogether, our results imply that mammalian GIIA sPLA2 may have a regulatory role in neuronal mitochondria and suggest a new line of study to further the understanding of its involvement in mitochondrial function and dysfunction.

The seminar will be held on-line on Thursday April 21, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

21. 4. 2022 Larisa Lara Popošek (PhD student at the Biotechnical faculty): The quest for new lipid-binding proteins and biotechnological applications

Abstract

Aegerolysins are relatively small proteins (15 kDa) with unique biotechnological potential. The main biochemical characteristic of aegerolysins is their high affinity for specific membrane lipids or lipid mixtures. In addition, they are non-toxic, and protocols for their isolation in recombinant form have already been developed. They can be found mainly in the kingdom of fungi and bacteria. In some production organisms, aegerolysins co-occur with MACPF (Membrane Attack Complex / Perforin) protein partners. Aegerolysins in combination with protein-protein form pores in target lipid membranes. The best studied aegerolysins and their protein partner are those from the fungal genus Pleurotus. Aegerolysins ostreolysin A (OlyA), ostreolysin A6 (OlyA6), pleurotolysin A (PlyA), and MACPF-containing pleurotolysin B (PlyB) were isolated from Pleurotus ostreatus, while aegerolysins erylysin A (EryA) and pleurotolysin A2 (PlyA2) and MACPF-containing erylysin B (EryB), were isolated from P. eryngii.
Aegerolysins are characterized by high affinity for the lipid receptor ceramide phosphoethanolamine (CPE), which is characteristic for some insects. In addition, some of these aegerolysins less specifically bind to the mixture of sphingomyelin (SM) and cholesterol (Hol), which is characteristic lipid mixture in vertebrate membranes. Fluorescently labeled aegerolysins can be therefore used to study these lipids, in the similar manner as antibodies are used to study the localization or presence of proteins in the cells. Although aegerolysins are non-toxic, they may, in the presence of a protein with the MACPF domain, become part of a cytolytic complex that perforates the membrane of those cells that contain the lipid receptor recognized by aegerolysin. Aegerolysins are therefore biocomponent cytolysins - component A (aegerolysin) recognizes the lipid target, and component B (protein partner with the MACPF domain) further allows the formation of pores in the target membrane.
Four novel aegerolysins, together with their native MACPF-containing protein partner from P. pulmonarius are currently being studied. So far, all five proteins have been isolated in recombinant form and the first experiments have been performed. You will therefore found out in the seminar: (i) whether aegerolysins from P. pulmunarious also recognize lipid receptors, (ii) whether do these aegerolysins in combination with MACPF protein partners show any hemolytic activity and whether they are (iii) insecticidal.
It is not surprising that mushrooms from the genus Pleurotus are not the only mushrooms that code for lipid-binding proteins. In the second part of the seminar we will introduce you with the surprising results which will show how many randomly harvested Slovenian wild mushrooms code for lipid-binding proteins and what are the lipid specificities of these unidentified proteins. Our main aim is to identify new proteins that bind to specific lipids and to prepare them in recombinant version. This would in turn allow as new biotechnological applications as it was previously shown for Pleurotus aegerolysins.

17. 3. 2022 Tadeja Bele (PhD student at the Jožef Stefan Institute and Biotechnical Faculty): Effects of selected α7-nicotinic acetylcholine receptor antagonists on lung cancer cells

Abstract

For decades, nicotinic acetylcholine receptors (nAChRs) were believed to be expressed only in central nervous system and at the neuro-muscular junctions. Then, they were detected also in lung cancer cells, where their elevated expression was linked to cancer development and progression by regulation of cell proliferation, cell cycle arrest and apoptosis. Their involvement in regulation of cell fate is not surprising considering the ubiquitous expression of nAChRs in mammalian cells and tissues, as well as their homologous ligand-gated ion channels (LGICs) in bacteria, algae and plants, which is implicating that they take part in a very conserved regulatory mechanism. Nicotine and its derivatives bind to nAChRs with higher affinity then their physiological ligand ACh and activate signalling pathways that trigger uncontrolled cell division, prevent apoptosis, cause angiogenesis and thus, support tumour growth and metastasis. Antagonists of nAChRs have shown a potential in cancer therapy in combination with already existing chemotherapeutics or ionizing radiation. The main goal of our study is to explore whether the binding of nAChRs antagonists to α7-nAChR, a receptor variant, which expressions has been found increased in lung cancer, can trigger cancer cell death. In addition, our interest is also analysis of signalling pathways that lead to this effect. Unfortunately, the majority of known naturally occurring nAChRs antagonists are different animal toxins, which, if used for cancer treatment, might induce serious side effects on normal tissue. Previously, it was shown that some secreted phospholipases A2 (sPLA2s) from snake venoms act as antagonists of nAChRs. They supress ACh-elicited ion currents. We prepared recombinant human orthologues of such snake venom sPLA2s, GV and GX sPLA2, to test whether they also act as nAChRs antagonists or not. In addition, by mutating the active site His48 into Gln (H48Q point mutation) we synthesized and fully characterized enzymatically inactive mutants of these two human sPLA2s. Electrophysiological studies on Xenopus laevis oocytes expressing α7-nAChR showed that all prepared sPLA2s were able to supress ACh-elicited ion currents thus, so they acted as α7-nAChR antagonists. Recombinant sPLA2s were further analysed on adult muscle type nAChR. Very interestingly, this revealed a possible selectivity of enzymatically inactive GV(H48Q) sPLA2 on α7-nAChR, exposing thus this sPLA2-ligand as the most promising in lung cancer treatment studies. Currently, we are studying effects of our recombinant human sPLA2s on various lung cancer cells in culture.

The seminar will be held on-line on Thursday March 17, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

17. 3. 2022 Veno Kononenko (PhD, researcher at the Biotechnical Faculty): Testing nAChR antagonists and nanodelivery systems for the development of a novel lung cancer treatment strategy

Abstract
Lung cancer remains one of the most commonly diagnosed cancer with a low survival rate. Smoking is a major risk factor for the development of lung cancer. The binding of nicotine to nicotinic acetylcholine receptors (nAChRs) on lung cancer cells activates various signaling pathways that promote lung cancer cell proliferation and prevent cancer cell apoptosis, which increases lung cancer invasiveness and cancer cell resistance to chemotherapeutics. A better understanding of the role of nAChR in lung cancer development and progression has raised the idea of using nAChR antagonists that would reverse the effects of nicotine and other nAChR agonists for therapeutic purposes. Many of the known antagonists do not act selectively on nAChR subtypes that are overexpressed in lung cancer cells (especially the α7-nAChR subtype), leading to adverse effects. To avoid such adverse effects, it is necessary to ensure that a particular antagonist acts predominantly on cancer cells. This can be achieved by using nanodelivery systems that are preferentially uptaken by cancerous cells. In our work, we investigate the in vitro effects of different nAChR antagonists on human lung adenocarcinoma cells (A549 cell line), which according to the literature express a large number of α7-nAChRs. The literature also shows that nicotine increases the proliferation of A549 cells. However, by labeling α7-nAChRs and fluorescence microscopy, we observed that our A549 cells do not express large number of α7-nAChRs. Our results also show no increased proliferation of A549 cells when exposed to nicotine. For this reason, we decided to test the effects of nicotine also on other lung and breast cancer cell lines (H23: human lung adenocarcinoma; H1688: human small-cell lung cancer; SK-MES: human squamous cell carcinoma; MDA; human breast cancer). We are using nAChR antagonists which are known to have anticancer activity (hexamethonium) and compounds that have the potential to act as nAChR antagonists based on their structure (various 3-alkylpyridinium salts: APS7, APS7-2, APS8-2, APS12-2, and APS12-3). In addition to the effects of free antagonist molecules, we are also investigating how nanodelivery systems prepared from gelatin nanoparticles with embedded antagonists affect the observed effects. Gelatin nanoparticles were synthesized by nanoprecipitation method, and we are investigating the concentration- and time-dependent influence of different nAChR antagonists (free and embedded in nanodelivery systems) on proliferation, viability, and apoptosis of different cancer cell lines. The anticancer effect of cisplatin (a commonly used chemotherapeutic drug in the treatment of lung cancer, to which cancer cells often become resistant) in combination with various nAChR antagonists (free and embedded in nanodelivery systems) is also being investigated.

17. 2. 2022 Ana Kump (PhD student at JSI): Lipid droplets modulate ferroptosis sensitivity in cancer cells

Abstract

Despite ongoing research and significant effort to develop effective therapies, cancer remains one of the leading causes of death worldwide. Numerous studies have suggested that triggering apoptosis is a promising way of reducing tumor growth, but this approach has not been particularly successful so far. However, recent studies have revealed that inducers of ferroptosis, a recently discovered type of non-apoptotic cell death, are more effective than conventional chemotherapy agents in killing some of the most aggressive types of cancer cells. Ferroptosis arises when the cellular redox protection systems fail to counteract the lethal damage induced by membrane lipid peroxidation, which in turn depends on membrane-resident polyunsaturated fatty acids (PUFAs), which are prone to oxidation. To reduce oxidized lipids, cells rely on two major anti-ferroptotic machineries, the glutathione peroxidase 4 (GPX4) pathway and the glutathione-independent ferroptosis suppression protein 1 (FSP1) system. Our recent studies demonstrated that when cancer cells are exposed to exogenous PUFAs, they sequester PUFAs into triglycerides stored within newly formed lipid droplets. Accordingly, inhibition lipid droplet biogenesis reduced lipid peroxidation, oxidative stress, and cell death. Here we asked whether lipid droplets modulate ferroptosis by storing and releasing PUFAs. We found that a combined inhibition of the GPX4 pathway and of lipid droplet biogenesis leads to induction of ferroptosis, but lipid droplets are not essential for FSP1-induced protection. Lipid droplets, on the contrary, play an important role in FSP1-mediated suppression of ferroptosis in lung cancer cells, which were resistant to GPX4-inhibition. Therefore, lipid droplet biogenesis affects ferroptotic sensitivity in both GPX4- and in FSP1-dependent cancer cells. Finally, inhibition of lipolytic breakdown of lipid droplets reduced lipid peroxidation and ferroptosis in response to GPX4 inhibition and PUFA overload in both breast and lung cancer cells. Similarly, inhibition of lipolysis contributed to increased survival and reduced lipid peroxidation levels in lung cancer cells with suppressed FSP1 system. Thus, the biogenesis and breakdown of lipid droplets determines the sensitivity of cancer cells to ferroptosis, most likely by regulating fatty acid trafficking and managing membrane lipid peroxidation.

The seminar will be held on-line on Thursday February 17, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

17. 2. 2022 Špela Koren (PhD student at JSI): Autophagy is involved in lipid droplet breakdown in serum-starved breast cancer cells

Abstract

Lipid droplets (LDs) are dynamic fat storage organelles present in most eukaryotic cells from yeast to men. LDs store triglycerides and cholesteryl esters and provide fatty acids for mitochondrial energy production, but also have many functions that extend beyond storage of energy-rich molecules. LDs accumulate in cells deprived of oxygen and nutrients, indicating their involvement in the cellular stress response. LDs are engaged in a complex relationship with autophagy, a major cellular recycling mechanism, which is activated during stress. Recent studies suggest that autophagy may participate in both LD biogenesis and breakdown, but LDs may also facilitate autophagy by providing lipids for autophagosomal membrane formation. It is not yet clear how this complex interplay is regulated and which molecular pathways are responsible for the execution of the multiple possible outcomes.
In this work, we asked if and how autophagy and LDs cooperate in the response of breast cancer cells to nutrient deficiency. We found that amino acid starvation, which is a physiological trigger for autophagy, promotes LD biogenesis in breast cancer cells. In contrast, during milder starvation in the absence of serum, LDs were broken down rapidly. Using live-cell confocal imaging, we show that LDs colocalize with autophagosomal and lysosomal structures within the first several hours of mild starvation, suggesting the involvement of “lipophagy”, an LD-selective form of autophagy, in the breakdown of LDs during serum starvation. In accordance, inhibition of lysosomal acid lipase (LAL), which is crucial for lysosomal hydrolysis of triglycerides, further elevated the colocalization between LDs and autophagosomes in serum-starved cells. Finally, silencing of the essential autophagic genes ATG5 and ULK1 augmented LD levels and reduced the colocalization of LDs with autophagosomes. These results suggest that autophagy is involved in the rapid breakdown of LDs following serum removal in aggressive breast cancer cells.

20. 1. 2022 Maja Hostnik (PhD student at the Biotechnical faculty): A method for targeting a specified segment of DNA to a bacterial microcompartment

Abstract

Bacterial microcompartments (MCPs) are protein organelles involved in various processes in cells. One of the largest MCPs are synthesized from the pdu operon and are involved in the 1,2-propanediol metabolism in a variety of bacterial taxa. Many metabolic enzymes which involve a specific peptide sequence at the amino terminus are incorporated into the lumen during the assembly of the MCP. These MCP packaging sequences can be linked to the protein of interest to direct it into the lumen of MCP.
In our study, we repurposed the Citrobacter freundii propanediol utilisation (Pdu) MCP to be used as a nucleic acid container. First, we constructed a plasmid carrying genes for shell proteins of the Pdu MCP and a transcription factor LacI labelled with the Pdu targeting peptide and the enhanced green fluorescent protein (EGFP). Since these genes were under pBAD promoter, we achieved self-assembly of the synthetic organelle containing fluorescently labelled LacI by adding arabinose to the transformed Escherichia coli cells. The fusion of the major Pdu subunit with the “Twin-Strep-tag” allowed us to isolate MCPs by affinity chromatography. The presence of LacI in the isolated MCPs was confirmed by fluorescence microscopy and mass spectrometry. Since LacI is a DNA-binding protein, we assumed that a specific DNA sequence could also be encapsulated within the microcompartment. Therefore, we introduced additional plasmid with multiple binding sites for LacI into E. coli cells carrying the plasmid with genes for assembly of Pdu MCP containing fluorescently labelled LacI. The DNA was indeed purified from isolated MCPs, and the results of high-throughput sequencing show that the selected DNA segment with the LacI target sites was successfully encapsulated within the MCP.
We anticipate that DNA could control the spatial organization of the enzymes in the MCP and such nucleocapsids can be used as a scaffold to obtain functional metabolic structures, which can increase metabolic yield and sequester toxic metabolic intermediates in the lumen of the MCPs.

The seminar will be held on-line on Thursday January 20, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

20. 1. 2022 Anja Pavlin (PhD student at the Biotechnical faculty): Small bacteriophage protein determines hierarchy over coresidential jumbo phage and influences its host biology

Abstract

Bacillus thuringiensis serovar israelensis is the biopesticide most widely used against insects including vectors of animal and human diseases. Among several extrachromosomal elements, this endospore-forming entomopathogen harbors two bacteriophages, a linear DNA replicon named GIL01, and a jumbo prophage known as pBtic235. Here we show that GIL01-encoded gp7, which interacts with the host LexA repressor, is a global transcription regulator and delays induction of pBtic235 after DNA damage, allowing GIL01 to selectively produce its own progeny. Moreover, acquired transcriptomes show that gp7 upregulates several virulence determinants, indicating the importance of gp7 in the pathogenesis of B. thuringiensis. Furthermore, our results imply that gp7 also increases the sporulation rate of its host, impacting an important survival strategy. We show that gp7 homologs, exclusively found in bacteriophages, act in a similar fashion to enhance LexA binding to DNA and are likely to also affect host gene expression. Our results provide evidence that GIL01 influences both its host and co-resident bacteriophages.

16. 12. 2021 Mauro Danielli (PhD, researcher at JSI): Interplay Between Autophagy and Lipolysis in Cancer Cells during Nutrient Stress

Abstract

Lipid droplets (LDs) are cytosolic fat storage organelles, which accumulate in stressed cells and in various pathologies, including cancer. Previous studies carried out in our laboratory have shown that LDs protect breast cancer cells from nutrient deprivation-induced stress, possibly by providing fatty acids (FAs) for essential cellular processes. Here, we investigated the relative importance of two major mechanisms of LD breakdown for cancer cell survival during starvation: lipolysis, catalyzed by neutral lipases, and lipophagy, an LD-specific form of autophagy.
Our results in serum-starved MDA-MB-231 human breast cancer cells reveal that silencing of adipose triglyceride lipase (ATGL), the major mammalian triglyceride lipase, compromises LD breakdown without affecting cancer cell death. However, ATGL-depleted cells displayed elevated autophagic flux, suggesting that autophagy/lipophagy could be activated as a compensatory mechanism for LD breakdown. To assess the importance of autophagy in LD breakdown, we silenced autophagy related 5 (ATG5), an essential protein involved in the formation of autophagic vesicles. siRNA-mediated knockdown of ATG5 reduced autophagic flux and increased apoptosis. Although LD content was not affected in ATG5-silenced cells, LD and lipolysis markers, including ATGL, were reduced. Finally, a combined suppression of lipolysis and autophagy in ATGL/ATG5 double knockdown cells partially reversed the effects of single ATG5 knockdown, suggesting that ATGL acts upstream of ATG5 during LD breakdown. In conclusion, our results suggest that lipolysis and lipophagy cooperate in lipid droplet breakdown in serum-starved cancer cells and that both contribute to cancer cell survival.

The seminar will be held on-line on Thursday December 16, starting at 12:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

16. 12. 2021 Maida Jusović (PhD student at JSI): Autophagy-Driven Lipid Droplet Formation Protects Cancer Cells from Nutrient Stress

Abstract

Lipid droplets (LDs) are organelles, which are emerging as central players in lipid metabolism in various cells exposed to stress. Accumulating evidence demonstrates a complex relationship between LDs and autophagy. Our primary aim is to define the role of autophagy in LD metabolism in cancer cells exposed to different types of nutrient stress. Our preliminary data shows that LDs are dynamically formed in HeLa cervical cancer cells depending on the severity and length of nutrient stress. Using inhibitors of diacylglycerol acyltransferase 1 and 2 enzymes (DGAT1 and DGAT2), which are involved in triglyceride biosynthesis, we show that LD biogenesis occurs under both severe and mild stress. We have also found that autophagic flux is elevated in starving HeLa cells and that inhibition of autophagy suppresses LD biogenesis. Importantly, both DGAT-mediated LD biogenesis and autophagy were essential for HeLa cell survival during severe starvation. However, during milder but prolonged serum-starvation DGAT-mediated LD biogenesis was not neccessary for HeLa cell survival, but compromising autophagy did induce cell death. Surprisingly, using live-cell confocal imaging we show that autophagosomal structures co-localize with LDs only when both lipolysis and autophagy are blocked, but not in the case of blocking only lipolysis. Altogether, our results suggest that autophagy drives LD biogenesis in HeLa cells exposed to both severe and mild starvation and that this relationship is essential for the protection of cancer cells against metabolic stress.

18. 11. 2021 Anastasija Panevska (PhD, researcher at BF): The story of ostreolysin A6 and what is next

Abstract

The aegerolysin from the oyster mushroom, ostreolysin A6 (OlyA6), interacts with the mammalian sphingomyelin in the presence of cholesterol, which regulates membrane properties and many intracellular signaling processes. In addition, the interaction of OlyA6 with membranes composed of cholesterol and ceramide phosphoethanolamine (CPE), a membrane lipid specific for invertebrates, especially insects, is increased 1000-fold. This lipid is found only in trace amounts or not at all in the membranes of higher organisms, including mammals. Ostreolysin A6, together with its partner protein pleurotolysin B (PlyB) produced by the same fungus, forms transmembrane pores or, when present in higher concentrations, can form crystal structures on the membrane surface that inhibit pore formation.
Recently, a single point mutation E69A was shown to abolish the ability of OlyA6 to recognize only sphingomyielin/cholesterol combinations in the membrane. Using the surface plasmon resonance method, we further investigated the interaction of the OlyA6 mutant with sphingomyielin only. In addition, we investigated whether OlyA6 can recognize a specific conformation of ceramide phosphoethanolamine in the membrane and whether this ability is lost by the E69A mutation. The OlyA6 E69A showed a 100-fold higher interaction with CPE membranes and could also recognize CPE-containing membranes lacking cholesterol. However, the lytic activity of OlyA6 E69A/PlyB was not altered compared with the activity of OlyA6/PlyB.
We also examined the interaction of OlyA6 with Balb 3T3 cells infected with cytomegalovirus and the fluidity of the lipid composition of these cells after infection. In addition, we examined internalization and explored the interaction of OlyA6 with late endosomes in Balb 3T3 and its colocalization with late endosome markers, suggesting a new potential application of this protein.

The seminar will be held on-line on Thursday November 18, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

18. 11. 2021 Neža Repar (PhD student at BF): The protective role of oleic acid is not related to its incorporation into lipid droplets in HUVEC cells treated with superparamagnetic iron oxide nanoparticles (SPIONs)

Abstract

Our study was based on the assumption that lipid metabolism and cell death are related. We investigated whether exogenous oleic acid (OA) enhances the ability of cells to cope with exposure to superparamagnetic iron oxide nanoparticles (SPIONs). Our hypothesis was that the addition of OA would protect cells from oxidative stress-induced cell death resulting from SPION exposure. We further hypothesized that OA would protect cells from SPION-induced cell death through incorporation into triacylglycerides (TAG) and formation of lipid droplets (LD), which could protect cells from the deleterious effects of SPIONs through various mechanisms. If the formation of LD is a mechanism of cell protection, we hypothesized that by preventing the incorporation of OA into LDs, OA would no longer protect against exposure to SPIONs. Our results showed that both N-acetylcysteine and alpha-tocopherol reduced the amount of ROS and decreased the cell death caused by SPIONs, suggesting that the cytotoxic effect of SPIONs is indeed related to oxidative stress. Pretreatment of HUVEC cells with OA also increased their ability to cope with the negative effects of SPIONs and was also associated with suppression of ROS formation. Surprisingly, the results showed that OA was able to suppress cell death induced by SPIONs even in the presence of DGAT inhibitors, implying that the formation of LD is not a prerequisite for exogenous OA to inhibit cell death induced by SPIONs. FTIR spectroscopy confirmed a severe effect of SPIONs at an exposure concentration of 50 μg/mL. SPIONs increased the amount of beta-sheets and random coil proteins, a phenomenon often associated with oxidative stress, as well as cell membrane morphology.

21. 10. 2021 Nina Zupanič (PhD, researcher at JSI): Advancements on the study of VaaSPH-1 towards development of the first intrinsic tenase inhibitor-based drug for treatment of venous thrombosis

Abstract

Preceding research focusing on the components of the nose-horned viper’s (Vipera ammodytes ammodytes) venom has identified a glycoprotein, named Vaa serine proteinase homolog 1 (VaaSPH-1), with a series of rather unique properties. The protein is an enzymatically inactive homologue of serine proteinases that significantly prolongs the activated partial thromboplastin time and therefore exhibits potent anticoagulant action in human blood. Its anticoagulant properties stem from its competitive binding to FVIIIa, while antagonizing the FIXa and thereafter inhibiting the formation of the intrinsic tenase complex. As currently used antithrombotic therapies are far from optimal and safe, development of the first intrinsic tenase inhibitor-based drug could attenuate thrombus formation and propagation without significantly increased risk of bleeding. To design and synthesize VaaSPH-1-based anticoagulants for preclinical studies, we must first define key structural properties important for its biological action. We have set up a mammalian cell line (HEK293F) that will enable production of recombinant VaaSPH-1 and its mutants, as well as FVIIIa A2 domain, in larger quantities required for experimental characterization of the interaction site between the two proteins. Concomitantly, we have isolated and purified more of the native VaaSPH-1 protein, for reference, but also for further research, focusing on its potential effects on other cellular structures (e.g. K+ channels). Based on the 3D model of the VaaSPH-1 and FVIIIa complex, our current goal is to design and synthesize low molecular mass anticoagulant candidates for subsequent evaluation of their effects on blood coagulation.

The seminar will be held on-line on Thursday October 21, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

21. 10. 2021 Anže Lovše (Master's degree student at the Biotechnical faculty): Small bacteriophage protein acts as a master regulator of expression in Bacillus thuringiensis serovar israelensis

Abstract

Bacillus thuringiensis serovar israelensis, an essential agent for the biological control of mosquitoes, carries many extrachromosomal elements. A linear molecule GIL01 and a circular plasmid pBtic235 correspond to bacteriophages that switch from the lysogenic to the lytic cycle in response to DNA damage. Using RNA sequencing and a newly developed pipeline for reproducible and transparent data analysis, we show global changes in the transcriptome after expression of a small protein gp7, encoded by GIL01, or after induction of lytic cycle of GIL01. The gp7 is a global transcription regulator and delays induction of the prophage pBtic235 after DNA damage, allowing GIL01 to preferentially produce its progeny. When we induced the lytic cycle of GIL01 we detected an upregulation of host SOS response and genes involved in genome maintenance over time, peaking 30 minutes after induction. In addition, we observed time- and induction-independent expression of structural and lytic genes of the GIL01 plasmid, suggesting additional regulatory mechanisms. The transcriptional patterns on the GIL01 indicate two functional modules that likely correspond to individual transcripts. Overall, this work provides the first characterisation of transcriptome dynamics in an important entomopathogenic strain of B. thuringiensis carrying GIL01 and pBtic235 plasmids.

23. 9. 2021 Kity Požek (M.Sc., JSI): Advancement on VaaMPIII-3, a disintegrin-like/cysteine-rich protein from the venom of Vipera a. ammodytes

Abstract

VaaMPIII-3 is a hemostatically active toxin found in the venom of the nose-horned viper (Vipera a. ammodytes) and represents a new P-IIIe subclass of snake venom metalloproteinases. It is a highly glycosylated 21 kDa protein consisting of two non-catalytic domains, the truncated disintegrin-like domain, and a cysteine-rich domain. We isolated VaaMPIII-3 from the venom of the nose-horned viper and determined its biochemical and functional properties. We found that it inhibits platelet aggregation induced by various agonists and thus contributes to the anticoagulant effect of the venom. As such, it could be an interesting starting molecule for the development of new antithrombotic drugs. We plan to determine its mechanism of action on blood clotting and investigate whether it has any other biological activities. Since VaaMPIII-3 is scarce in the venom, our aim was to produce it recombinantly and verify its conformity with the natural form. We expressed it in E. coli in fusion with a maltose-binding protein (MBP) tag, which enabled its efficient purification from bacterial lysate by amylose-affinity chromatography. After proteolytic removal of the MBP tag, we purified the recombinant VaaMPIII-3 by RP-HPLC under alkaline conditions. We confirmed that it is suitable for use in place of natural VaaMPIII-3 for further studies of its biological functions and underlying mechanisms, which could lead to the development of new antithrombotic drugs.

The seminar will be held on-line on Thursday September 23, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

23. 9. 2021 Aljoša Marinko (Master's degree student at JSI): Recombinant protein expression of VaaCRISP-1, a cysteine-rich secretory protein isoform from the venom of nose-horned viper

Abstract

Cysteine-rich secretory proteins (CRISPs) are found throughout the animal kingdom exerting different functions. They are monomeric proteins with a molar mass of 20 to 30 kDa. Most CRISPs found in snake venoms are ion channel inhibitors, including voltage-gated K+ channels, Ca2+-dependent K+ channels, voltage-gated Ca2+ channels and ion channels regulated by cyclic nucleotides. The biological role of CRISPs from the venom of the nose-horned viper (Vipera ammodytes ammodytes, Vaa) is not clear yet. To study their function more thoroughly, we decided to prepare recombinant VaaCRISP-1 (rVaaCRISP-1). cDNA sequence of rVaaCRISP-1 was inserted into the vector pMAL-c5x. The construct was inserted into the bacteria E. coli, strain ER2523. Fusion protein consisted of the N-terminal maltose binding protein (MBP) that enabled the isolation process of rVaaCRISP-1 on an amylose resin and a linker containing the Factor Xa (FXa) cleavage site. Expression of pMAL-CRISP on a large scale (2 L Erlenmeyer flasks) failed. Namely, the analysis of bound fraction after affinity chromatography on an amylose column showed only large amounts of MBP-tag and no fusion protein. On the other hand, expression on a smaller scale (250 mL Erlenmeyer flasks) was successful as we isolated pMAL-CRISP. After cleavage of the fusion protein with FXa a small amount of rVaaCRISP-1 was purified by RP-HPLC on a C18 column. Overall, our results show that the employed expression system is not optimal for the production of rVaaCRISP-1. Therefore, we are exploring other strains of E. coli and other types of expression cells (e.g., mammalian cells) to achieve more efficient recombinant protein expression.

17. 6. 2021 Mojca Dobaja Borak (PhD student at MF and JSI): Isolation and characterization of proteins from Vipera ammodytes ammodytes (Vaa) venom that induce transient and reversible thrombocytopenia of functional platelets

Abstract

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 agents used to prevent and reverse platelet aggregation in acute coronary disease, acting as GPIIb/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). In our hospital practice, thrombocytopenia of functional platelets without bleeding was observed in patients bitten by Vaa. C-type lectin-like proteins (snaclecs) are the heterodimeric proteins found in the venom of many species of the Viperidae family. They are interfering with haemostasis by acting on platelet receptors. Based on preliminary data from in vitro studies, we predict their binding to the GPIb receptors, thereby preventing interaction with the von Willebrand factor and inhibiting the process of platelet adhesion to subendothelial collagen in vivo.
We present preliminary results of purification and characterization of snaclecs isolated from Vaa venom. Flow cytometry results showed a specific interaction between isolated Vaa snaclecs and platelet GPIb receptor without expression of GPIIb/IIa and P-selectin, the markers of platelet activation. This is consistent with the results of flow cytometry of platelets from Vaa-bitten patients.

The seminar will be held on-line on Thursday June 17, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

17. 6. 2021 Gašper Žun (PhD student at BF and JSI): Finding sources and their inheritance of salt tolerance as a polygenic trait

Abstract

Polygenic trait’s genetic architecture is complex and all its causative elements are generally unknown. To dissect and assess causative variants for NaCl tolerance as a case of a polygenic trait, we crossed 2 parental haploid yeast strains of large phenotypic difference. We monitored the distribution of the phenotype in a population of their progeny. After each phenotyping step of a given generation, we selected the offspring strain with the most advanced phenotype.
Analysis of distribution of parental alleles in populations growing in extreme saline and reference conditions allowed us to predict potential causative quantitative trait loci (QTL). On the basis of genomic sequences of the selected segregants we followed the inheritance of predicted quantitative trait loci over the generations. Inheritance of potential quantitative trait loci, average population phenotype shift, quantitatively assessed phenotype of selected segregants and their isogenic derivatives with swapped alleles of possible quantitative trait genes (QTG) pointed out importance of certain quantitative trait elements for salt tolerance in yeast.

20. 5. 2021 Adrijan Ivanušec (PhD student at JSI): Interaction between secreted phospholipases A2 and mitochondria

Abstract

Group IIA secreted phospholipase A2 (GIIA sPLA2) is a mammalian orthologue of ammodytoxin (Atx), a β-neurotoxic GIIA from the snake venom. It plays both physiological and pathophysiological roles in mammalian brain. Physiologically, it is involved in the regulation of neurotransmission, neuritogenesis and mitochondrial homeostasis, while pathologically, it is implicated in neurodegenerative and cerebrovascular diseases. Both β-neurotoxic and endogenous GIIA sPLA2s inflict damage to neuronal mitochondria in pathological conditions. However, the molecular mechanisms of action of sPLA2s in these pathologies are not yet clear. To further dissect the interaction between GIIA sPLA2s and mitochondria, we prepared the recombinant rat GIIA sPLA2 and its enzymatically inactive mutant and evaluated their effect on neuronal mitochondria. Heterologous competition assay using radioiodinated Atx showed that the mammalian GIIA sPLA2 binds to the subunit II of cytochrome c oxidase (CCOX-II), an essential constituent of the respiratory chain complex, previously identified as the mitochondrial receptor of Atx. The determined binding affinity of GIIA sPLA2 was in the μM range, 100-fold lower than that of Atx. However, homologous competition assay using radioiodinated GIIA sPLA2 revealed that the endogenous sPLA2 primarily targeted another binding protein in mitochondria, which has an apparent mass of around 20 kDa (R20). Interestingly, GIIA sPLA2 and its mutant substantially inhibited CCOX activity in isolated rat mitochondria, suggesting that R20, a newly detected mitochondrial receptor for GIIA, is a subunit of CCOX. On the other hand, they only had a subtle effect on CCOX activity in rat brain tissue sections and PC12 cells, as determined by histochemical staining and flow cytometry, respectively. Taken together, our results indicate a possible regulatory role of the endogenous GIIA sPLA2 in mitochondria, opening an important direction of study to advance the understanding of the involvement of the mammalian GIIA sPLA2 in mitochondrial function and dysfunction.

The seminar will be held on-line on Thursday May 20, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

20. 5. 2021 Ana Kump (PhD student at JSI): Lipid droplets – antioxidant organelles that protect cancer cells from ferroptosis

Abstract

Despite ongoing studies and a lot of effort to develop effective therapies, cancer remains one of the most common causes of death worldwide. In recent therapeutic approaches, activating apoptosis has been suggested as a promising way of inducing cancer cell death, but these have so far not been highly successful. However, latest studies have reported that inducers of ferroptosis, a recently discovered form of programmed cell death, are more effective in killing the some of the most aggressive types of cancer cells. Ferroptosis arises when the cellular redox protection systems are unable to counteract the lethal damage induced by membrane lipid peroxidation. In the core of the mechanisms of ferroptosis are membrane-resident polyunsaturated fatty acids (PUFAs), which are prone to oxidation. Cells rely on two major anti-ferroptotic machineries to reduce oxidized lipids, the glutathione peroxidase 4 (GPX4) pathway and the glutathione-independent ferroptosis suppression protein 1 (FSP1) system. Our recent studies revealed that when cancer cells are exposed to exogenous PUFAs, they sequester PUFAs into triglycerides stored within newly formed lipid droplets. Accordingly, inhibition of triglyceride synthesis and lipid droplet biogenesis reduces lipid peroxidation, oxidative stress and cell death. We thus asked whether lipid droplets are modulators of ferroptotic cells death. We found that a combined inhibition of the GPX4 pathway and of lipid droplet biogenesis leads to induction of ferroptosis in aggressive breast cancer cells, but lipid droplets are not required for FSP1-induced protection. On the contrary, in lung cancer cells, which were resistant to GPX4-inhibition, lipid droplets play an important role in FSP1-mediated suppression of ferroptosis. Therefore, lipid droplet biogenesis modulates ferroptotic sensitivity in both GPX4- and in FSP1-dependent cancer cells. Finally, blocking the lipolytic breakdown of lipid droplets reduced lipid peroxidation and ferroptosis upon GPX4 inhibition, suggesting that lipid droplets both sequester and release oxidized lipids. Our results demonstrate that lipid droplets are antioxidant organelles, whose synthesis and breakdown determines the sensitivity of cancer cells to ferroptosis, likely by regulating fatty acid trafficking and managing membrane lipid peroxidation.

22. 4. 2021 Tadeja Bele (PhD student at JSI and the Biotechnical Faculty): Effects of selected α7-nicotinic acetylcholine receptor antagonists on adenocarcinoma cancer cells

Abstract

Nicotinic acetylcholine receptors (nAChRs) have long been regarded solely as mediators of neurotransmission, but their wide distribution in human body is suggesting a much more versatile physiological role. Moreover, in some types of cancer, for example lung cancer, which represents the leading cause of cancer death according to the American Cancer Society, the expression of nAChRs has been found elevated. Moreover, nAChRs have been associated with regulation of cell proliferation, apoptosis, angiogenesis and epithelial to mesenchymal transition. It has been observed that binding of nicotine, its derivatives and other agonists of nAChRs to specific subtypes of nAChRs (mainly α7 and α9) increased intracellular Ca2+ levels. Furthermore, α7-nAChRs agonists also activated signalling pathways that can trigger uncontrolled cell division, prevent apoptosis, cause angiogenesis and thus support tumour growth and metastasis. On the contrary, antagonists of these receptors have shown opposite effects, suggesting their potential usefulness in cancer therapy. The main goal of our study is to explore whether the binding of nAChRs antagonists to α7-nAChRs can trigger cell death and to analyse signalling pathways that lead to this effect. However, the majority of known naturally occurring nAChRs antagonists are different animal toxins, which, if used for cancer treatment, would also have side effects on normal tissue. Vulfius et al. (2014) showed that secreted phospholipases A2 (sPLA2s), isolated from snake venoms from families Elapidae and Viperidae, act as antagonists of nAChRs by supressing ACh-elicited ion currents. In the initial phase of our study, we prepared their recombinant human orthologues, sPLA2 group V and X sPLA2s and their enzymatically inactive mutants with a single point mutation in their active site. Currently, we started with electrophysiological studies of their activity on α7-nAChRs expressed in Xenopus laevis oocytes and in vitro studies of their effect on adenocarcinoma cell line A549. Therefore, we will also use a mutated form of α-conotoxin ArlB isolated from Conus arenatus, which showed a selective binding to α7-nAChRs.

The seminar will be held on-line on Thursday April 22, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

22. 4. 2021 Veno Kononenko (researcher at the Biotechnical Faculty): Nanodelivery systems of nAChR antagonists for the development of a novel lung cancer treatment strategy

Abstract

Major problems in the treatment of lung cancer are the indiscriminate action of drugs on healthy cells and cancer resistance to chemotherapy. Smoking is a major risk factor for the development of lung cancer. The binding of nicotine to nicotinic acetylcholine receptors (nAChRs) on lung cancer cells activates various signaling pathways that promote lung cancer cell proliferation and prevent cancer cell apoptosis, which increases lung cancer invasiveness and cancer cell resistance to chemotherapeutics. A better understanding of the role of nAChR in lung cancer development and progression has raised the idea of using nAChR antagonists that would reverse the effects of nicotine and other nAChR agonists for therapeutic purposes. Many of the known antagonists do not act selectively on nAChR subtypes that are overexpressed in lung cancer cells (especially the α7-nAChR subtype), leading to adverse effects. To avoid such adverse effects, it is necessary to ensure that a given antagonist acts predominantly on cancer cells. This can be achieved by using nanodelivery systems that are preferentially uptaken by cancerous cells.
In our work, we investigate the in vitro effects of different nAChR antagonists on human lung adenocarcinoma cells (cell line A549), which express a large number of α7-nAChRs. The effect of nAChR antagonists will also be tested on human colon cancer cells (cell line HT29), which express a very low number of α7-nAChRs compared to A549 cells. We use both nAChR antagonists which are known to have anticancer activity (hexamethonium) and compounds that have the potential to act as nAChR antagonists based on their structure (various 3-alkylpyridinium salts: APS7, APS7-2, APS8-2, APS12-2, and APS12-3). In addition to the effects of free antagonist molecules, we will also investigate how nanodelivery systems prepared from gelatin nanoparticles with embedded antagonists affect the observed effects. We will investigate the concentration- and time-dependent influence of various nAChR antagonists (free and embedded in nanodelivery systems) on the proliferation, viability, and apoptosis of A549 and HT29 cells. The anticancer efficacy of cisplatin (a commonly used chemotherapeutic drug in the treatment of lung cancer, to which cancer cells often become resistant) in combination with various nAChR antagonists (free and embedded in nanodelivery systems) will also be evaluated. In addition, we will evaluate how a chemotherapeutic (cisplatin), a nAChR agonist (nicotine) and nAChR antagonists influence the activity of enzymes involved in the synthesis and degradation of ACh, which acts as an autocrine growth factor for lung cancer cells. Our results will provide new insights into the effect of nAChR antagonists on lung cancer cells and the development of nanodelivery systems for lung cancer treatment.

18. 3. 2021 Anja Pavlin (PhD student at the Biotechnical Faculty): Small proteins modulating the LexA-like regulators of the bacterial SOS response

Abstract

The SOS response is a highly conserved mechanism by which bacteria detect and repair DNA damage within the cell. LexA, the master repressor of the SOS response, directly regulates expression of not only the genes involved in the repair or replication of damaged DNA but also genes involved in the movement of mobile elements, the propagation of virulence factors and the lytic cycle of temperate phages. Tectivirus GIL01 infects the Gram- positive bacterium Bacillus thuringiensis that unlike other temperate phages uses host's repressor of the SOS response, LexA, to maintain its lysogenic state. LexA efficiently represses bacteriophage transcription only if the small phage-encoded protein gp7 is also present. Gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites. This was the first report of an accessory factor interacting with LexA to regulate transcription.
To elucidate whether this mechanism is unique only to GIL01 we screened 13 small protein candidates from tectiviruses of the Bacillus cereus sensu lato group, bacteria Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii (a known human pathogen) for possible interaction with LexA-like repressors. We used different approaches to identify the possible gp7-like small proteins: amino acid sequence homology, structural homology, identification of possible LexA protein partners with pull-down assays followed by identification with mass spectrometry, and gene position and transcriptional direction relative to genes of LexA regulators. We examined the direct interaction by surface plasmon resonance and observed a high affinity interaction, similar to gp7, of all tectiviral gp7 homologs and the structural homolog M1QNS5 with LexA repressors from Gram-positive B. thuringiensis and S. aureus. No significant direct interaction was observed for other small proteins and selected LexA-like ligands. We also observed that tectiviral gp7 homologs enhance LexA DNA binding in a manner similar to gp7. We confirmed a direct interaction of DdrR with UmuDAb (LexA-like repressor in A. baumannii), its C-terminal domain, and UmuD, a component of DNA polymerase V. Our results imply that proteins supporting the action of LexA-like transcription factors are common to many bacteria.
In addition, we unravel the effect of gp7 on the B. thuringiensis transcriptome. We observed that the presence of gp7 prevents the activation of multiple host and phage-encoded genes. Remarkably, gp7 represses the induction of the lytic cycle of the secondary bacteriophage pBtic235, making GIL01 the primary phage able to exhaust the host for the production of its own new virions.

The seminar will be held on-line on Thursday March 18, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

18. 3. 2021 Neža Repar (PhD student at the Biotechnical Faculty): (Exogenous) oleic acid suppresses superparamagnetic iron oxide nanoparticle-induced ferroptosis (in human umbilical vein endothelial cells)

Abstract

Ferroptosis is an iron-mediated form of programmed cell death first proposed by Dixon et al. in 2012. It is characterized by increased intracellular iron levels and inactivation of phospholipid hydroperoxide glutathione peroxidase 4 (GPX4), followed by accumulation of lipid peroxides. Ferroptosis could not be prevented by chemical or genetic inhibitors of apoptosis or inhibitors of necroptosis, indicating that its mechanism is unique. Ferroptosis is also morphologically distinct from other types of cell death and manifests mainly as shrinkage of mitochondria with increased membrane density and reduction of mitochondrial cristae.
The aim of our work was to elucidate the role of lipid peroxidation (ferroptosis) in cell death induced by superparamagnetic iron oxide nanoparticles (SPIONs). Non-cancerous human umbilical vein endothelial cells (HUVEC) were used as a cell model. Endothelial cells are not highly susceptible to ferroptosis; however, they are among the first cells to come into contact with nanoparticles when administered intravenously.
We found that SPIONs induce increases in reactive oxygen species (ROS), lipid peroxidation, and cell death in a concentration-dependent manner. We further tested whether ferrostatin-1 and α-tocopherol, inhibitors of ferroptosis, could block cell death induced by SPIONs. Treatment with either inhibitor simultaneously with SPIONs reduced the amount of ROS, lipid peroxidation, and cell death. These results suggest that ferroptosis plays an important role in SPIONs-induced cell death.
Since it was previously discovered that exogenous oleic acid (OA) inhibits the induction of ferroptosis in human fibrosarcoma cells HT -1080, we aimed to investigate whether OA can also decrease the level of ferroptosis in human endothelial cells exposed to SPIONs and whether lipid droplets are involved in the protective role of OA. Our results showed that pretreatment of HUVEC cells with OA increased their resistance to cell death induced by SPIONs. This protective effect was associated with the suppression of ROS accumulation and lipid peroxidation.
A study by Magtanong et al. (2019) showed that the protective effect of monounsaturated fatty acids (FA) against ferroptosis does not depend on the formation of lipid droplets, but on the displacement of easily oxidizable polyunsaturated fatty acids (PUFA) from membrane phospholipids and the concomitant suppression of membrane lipid peroxidation. We therefore hypothesized that triacylglycerol synthesis and lipid droplet formation are also not necessary for exogenous OA to inhibit ferroptosis caused by SPIONs in endothelial cells. Using DGAT1- and DGAT2-specific inhibitors T863 and PF -06424439, respectively, we blocked neutral lipid synthesis and lipid droplet formation in HUVEC cells. Our results showed that 30 μM OA in the presence of DGAT inhibitors decreased cell death induced by SPIONs. Our results are consistent with the findings of a study by Magtanong et al. and suggest that the incorporation of oleic acid into lipid droplets is not critical for its protection against ferroptosis.

18.2. 2021 Andreja Habič (Master’s degree student at FCCT): A Novel Method for Discovery of DNA–Protein Interaction Partners

Abstract

DNA–protein interactions govern many cellular processes, including precise temporal regulation of transcription. Discovery of DNA regulatory elements and DNA-binding transcription factors, as well as understanding of their interactions, are of great importance for they provide insight into the development of many diseases, allowing the design of new treatments. Methods for detection of protein interaction partners of a known DNA are suitable for the detection of strong and stable interactions, however, weak and transient ones are often overlooked. To be able to detect all types of interactions, we set on to develop a novel approach for DNA–protein partners discovery, which is based on a three-component system that enables enzyme-catalysed proximity labeling of proteins with biotin. The system consists of a nucleotide sequence F-DNA and two chimeric proteins, Tus-TurboID and GAL4(1–147)-sfGFP. F-DNA contains DNA sequence of interest and binding sites for chimeric proteins, so the components of the system and target interaction partners are colocalised. Tus-TurboID contains a mutant biotin ligase which biotinylates colocalised proteins on F-DNA, whereas GAL4(1–147)-sfGFP represents the internal control of the system’s functionality. The system can be used either in vivo or in vitro. As a proof-of-concept we prepared an in vitro system involving a known DNA–protein complex, GADD–p53, which is now in the process of optimisation.

The seminar will be held on-line on Thursday February 18, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

21.1. 2021 Maida Jusović (PhD student at Jožef Stefan International Postgraduate School): Autophagy drives lipid droplet formation in severely starved cancer cells

Abstract

The seminar will be held on-line on Thursday January 21, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

21.1. 2021 Špela Koren (Master’s degree student at FCCT): Autophagy is involved in lipid droplet breakdown in serum-starved breast cancer cells

Abstract

Lipid droplets (LDs) are dynamic and multifunctional fat storage organelles present in most eukaryotic cells from yeast to men. LDs have many functions beyond storage of energy-rich molecules and are involved in the regulation of lipid metabolism, cell signaling and protein trafficking. LDs accumulate in cells deprived of oxygen and nutrients, indicating their involvement in the cellular stress response. Recent studies have shown that LDs are engaged in a complex relationship with autophagy, the major cellular recycling and stress response pathway. Autophagy may participate in both LD biogenesis and breakdown, whereas LDs may provide essential lipids for the initiation of autophagy. It is not yet clear how this complex interplay is regulated and which molecular pathways are responsible for the execution of the multiple possible outcomes. These questions are particularly important in cancer cells, which are often exposed to nutrient and oxygen fluctuations in the inhospitable tumour microenvironment and use both autophagy and LDs for protection against metabolic stress.
In this work, we aim to determine if and how autophagy and LDs cooperate in the protection of breast cancer cells against starvation. We found that LD biogenesis is upregulated during severe amino acid deprivation in Hanks' balanced salt solution, whereas milder starvation in the absence of serum induces LD breakdown. Intriguingly, we found that autophagy is active during both severe and mild starvation. Using live-cell confocal imaging we show that autophagosomal and lysosomal structures mostly colocalize with LDs during mild, but not during severe starvation, suggesting the involvement of “lipophagy”, an LD-selective form of autophagy, in starvation-induced breakdown of LDs. In accordance, inhibition of autophagic flux with bafilomycin A1 or inhibition of lysosomal acid lipase (LAL), which is crucial for lysosomal hydrolysis of triglycerides, further elevated the colocalization between LDs and autophagosomes. These results suggest that autophagy is active also during mild starvation and is involved in the breakdown of LDs in aggressive breast cancer cells.

17. 12. 2020 Maja Hostnik (BF): Isolation of a bacterial microcompartment organelle containing a selected DNA

Abstract

Bacterial microcompartments (BMCs) are protein organelles involved in various processes in cells. One of the largest BMCs are synthesized from the Pdu operon and are involved in the 1,2-propanediol metabolism of Salmonella enterica. These BMCs consist of 8 different Pdu subunits, which first assemble themselves into homo-hexamers or homo-pentamers and then form microcompartments. Several enzymes are selectively encapsulated in BMCs via their specific peptide carried by the enzyme. A heterologous cargo can also be encapsulated within the empty Pdu compartment by fusing the desired proteins with the signal sequences of the native Pdu enzymes.
The aim of our work was to design a program inside the bacterium to package a specific DNA fragment into the lumen of the Pdu shell. First, we constructed a plasmid carrying genes necessary for the assembly of the Pdu microcompartment and the transcription factor LacI labelled with the Pdu targeting peptide and the enhanced green fluorescent protein (EGFP). Since these genes were under pBAD promoter, we achieved self-assembly of the synthetic organelle containing fluorescently labelled LacI by adding arabinose to the transformed Escherichia coli cells. The fusion of one of the Pdu subunits with the Twin-Strep-tag later allowed us to isolate microcompartments by affinity chromatography. We confirmed the presence of LacI in the Pdu lumen by fluorescence microscopy, negative stain transmission electron microscopy and mass spectrometry. Since LacI is a DNA-binding protein, we assumed that a specific DNA sequence could also be encapsulated within the microcompartment. Therefore, we introduced additional plasmid with binding sites for LacI into E. coli cells carrying the plasmid with genes for Pdu compartment and fluorescently labelled LacI. The DNA was indeed purified from isolated microcompartments, and the results of high-throughput sequencing show that the selected DNA segment with the LacI target sites was successfully encapsulated within the microcompartment and protected from DNase treatment.

The seminar will be held on-line on Thursday December 17, starting at 14:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

17. 12. 2020 Matej Skočaj (BF): Aegerolysin-like proteins as new theranostic tools in periodontal disease and prosthetic rehabilitation

Abstract

Periodontal diseases (PD) are initiated by periodontal pathogens which can be found in bacterial plaque, complex biofilms attached to the tooth surface. The most studied periodontal pathogens are Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Aggregatibacter actinomycetemcomitans and some others. Their presence and/or quantity strongly relates to the initiation of PD, disease progression, and unsuccessful periodontal therapies. In prosthetic treated teeth, the biofilm attachment depends on surface roughness of prosthetic restorative materials, free surface energy of the materials and their surface characteristics. There is a higher plaque retention on the surface of polymer materials in comparison to metal and ceramic materials, which may accelerate the course of periodontitis in high risk patients.
Currently, diagnosis of PD is primarily based upon an assessment of the already apparent tissue breakdown (e.g. pocket probing depth, clinical attachment loss), which hampers early detection of the disease and after treatment follow-up. One of the potential molecules that can be considered as a biomarker for early detection of PD is a specific sphingolipid molecule, ceramide phosphoethanolamine (CPE), as well as its dihydrogenated form, dihydroceramide phosphoethanolamine (DCPE), since they are the major sphingolipids in cell membranes of Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia. In the membranes of vertebrate cells and Gram-positive bacteria CPE and DCPE are present only in minute amounts.
In the past years, an increasing number of lipid-binding proteins have been developed as probes for detecting and visualising the distribution of specific lipid species. In this respect, aegerolysins can be utilised as efficient molecular tools for detecting and visualizing CPE. Aegerolysin erylysin A (EryA) strongly associate with CPE/Chol-containing membranes, and show pore-forming activity, when combined with pleurotolysin B (PlyB), a 59-kDa protein partner with a membrane-attack-complex/perforin (MACPF). Therefore, we propose the use of CPE-sensing aegerolysin EryA for detection of PD and the combination of CPE-sensing EryA and MACPF domain containing PlyB for specific eradication of CPE-containing periodontal bacteria. In our preliminary results we have confirmed that the fluorescently labelled aegerolysin EryA (EryA-mCherry) strongly binds to the lipid mixtures composed of CPE/Chol and to the total lipids extracted from Porphyromonas gingivalis. Using calcein release assay, we have further demonstrated that the mixture of EryA and pleurotolysin B (PlyB), a MACPF domain containing protein, permeabilized CPE-containing membranes. We also have shown the presence of CPE and DCPE in all samples of periodontitis patients, and in only 20% collected samples from young healthy individuals. Thus, the main objectives of the proposed project are: 1. analysis of interactions between aegerolysins and lipids/lipid mixtures using biophysical methods (TLC/dot blot, sedimentation assay, surface plasmon resonance, calcein release assay), 2. analysis of aegerolysin binding to CPE/DCPE containing bacteria associated with periodontal disease using scanning electron microscopy (HR-SEM), 3. the use of aegerolysins and their fluorescently labelled variants for the detection of CPE and DCPE in dental plaque and saliva samples of periodontitis and prosthetically rehabilitated patients and 4. verification of the antibacterial potential of selected aegerolysin EryA combined with their partnering proteins with the MACPF domain (e.g., PlyB, EryB) against periodontal pathogens.

17. 11. 2020 Kity Požek (Bolonian 2nd degree student at FCCT and JSI): Characterization of VaaMPIII-3, a snake venom protein that defined a novel P-IIIe subclass of snake venom metalloproteinases

Abstract

Snake venom metalloproteinases (SVMPs) are structurally and functionally diverse family of toxins that are abundant in viperid venoms and have evolved from ADAM28 (a disintegrin and metalloproteinase) protein. They typically affect hemostasis and relevant tissues and cause hemorrhage. P-III class SVMPs consist of a catalytic domain, metalloproteinase (MP), and non-catalytic, disintegrin-like (D) and cysteine-rich (C), domains. SVMPs consisting only of non-catalytic domains have been found in viperid venoms before, but they have been regarded as products of P-III SVMP autolysis. Our research group has recently discovered such SVMP called VaaMPIII-3 in Vipera ammodytes ammodytes (Vaa) venom, which, however, results from an evolutionary MP-domain loss. This finding was the basis for introduction of a new SVMP subclass, P-IIIe. In this work, we have isolated VaaMPIII-3 from the Vaa venom and characterized it biochemically and functionally. We found that VaaMPIII-3, which consists of a partial D-domain and a C-domain, is an acidic glycoprotein of about 21 kDa of which 4 kDa corresponded to Asn-bound sugars. In accordance with its primary structure, we confirmed that the molecule has one free Cys residue, predominantly at the position 6, which is likely implicated in formation of a covalent dimer. VaaMPIII-3 inhibited ADP-, collagen- and arachidonic acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination. As such, it represents a suitable basis for development of new antithrombotics. To further investigate the structural determinants underlying the biological functions of VaaMPIII-3, we prepared a model of its three-dimensional structure. We also prepared the recombinant form of the protein and demonstrated its identity with the natural form. Full understanding of this non-catalytic venom protein, which is our goal, will certainly help to decipher physiological roles of its human orthologues - ADAMs.

The seminar will be held on-line on Tuesday November 17, starting at 15:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

17. 11. 2020 Tihomir Rubil (Bolonian 2nd degree student at BF and JSI): Isolation of genomic DNA from the nose-horned viper and initial analysis of its metalloproteinase genes

Abstract

Snake venoms, including that of the nose-horned viper (Vipera ammodytes ammodytes, Vaa), are a valuable source of pharmacologically interesting substances. Of particular interest for this thesis were snake venom metalloproteinases (SVMPs). These proteins are mainly responsible for hemorrhagic and hemostatic effects of snake venom. Of notable reference are metalloproteinases that belong to the P-III (MPIII) class. These metalloproteinases are diverse in both quaternary protein structure as well as in their function. The organization and features of their genes are yet largely unknown. The aim of this thesis was to isolate Vaa genomic DNA, and to amplify and analyze genomic sequences encoding two metalloproteinases, Vaa hemorrhagin 4-A and Vaa metalloproteinase III-like protein 3 (Vaa-MPIII-3). Their presumed nucleotide sequences were amplified by PCR and determined by the Sanger dideoxy-chain termination method. Results indicated that the specific amplification attempts for both genomic sequences were unsuccessful. In addition, a putative contig of 23,133 bp harboring the genomic sequence coding for Vaa-MPIII-3, obtained from the whole Vaa genome sequencing project, was acquired and its structure analyzed. The Vaa-MPIII-3 gene is composed of 10 exons and 9 introns. It has been confirmed that Vaa-MPIII-3 represents a new subtype of metalloproteinases lacking the entire metalloproteinase (enzymatic) domain and the first part of the succeeding disintegrin domain, reflecting a unique evolution of its gene, separated from that of enzymatically active snake venom MPIII.

20. 10. 2020 Anastasia Panevska (BF): Interactions of aegerolysins from fungal genus pleurotus with artificial and biological membranes

Abstract

Aegerolysins from the oyster mushroom ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) interact with the ceramide phosphoethanolamine (CPE), a membrane lipid specific for invertebrates, especially insects. This lipid occurs only in trace amounts or is completely absent in the membranes of higher organisms, including mammals. Aegerolysins form transmembrane pores together with their partner protein pleurotolysin B (PlyB), which is produced by the same fungus. On the other hand, agerolysins in higher concentrations can form crystal structures on the membrane surface that inhibit pore formation. Aegerolysin binds to artificial and biological membranes containing very small amounts of CPE (1 mol %). This interaction is crucial for the development of novel biopesticides that target lipid rather than protein receptors. In the presence of PlyB, aegerolysin proteins have a toxic effect on insects containing the lipid receptor in their membranes. Such protein complexes show a toxic effect against the larvae of the two economically most important plant pests - the Western Corn rootworm and the Colorado potato beetle. The toxicity of these protein complexes is comparable to the toxicity of protein-containing crystal toxins (Cry proteins). These proteins could be used as biopesticides to control the Colorado potato beetle and the Colorado potato beetle and the rootworm Western Corn.

The seminar will be held on-line on Tuesday October 20, starting at 15:00. Non-members of our Programme Group, who would like to attend the seminar contact Igor Križaj (igor.krizaj@ijs.si) to receive a link.

13. 2. 2020 Adrijan Ivanušec (JSI): Interaction between secreted phospholipases A2 and mitochondria

Abstract

Group IIA secreted phospholipase A2 (GIIA) is a mammalian orthologue of ammodytoxin (Atx), a β- neurotoxic GIIA (β-ntxs) from the snake venom. It plays both physiological and pathophysiological roles in mammalian brain. Physiologically, it is involved in regulation of neurotransmission, neuritogenesis and mitochondrial homeostasis, while pathologically, it is implicated in neurodegenerative and cerebrovascular diseases. The action of GIIA in these pathologies is far from being clear on the molecular level. To reveal them, β-ntxs may be very useful (tools) as they inflict apparently identical damage to neuronal mitochondria as GIIA. Previously, we detected a high affinity membrane receptor of Atx in neuronal mitochondria and identified it as the subunit II of cytochrome c oxidase (CCOX-II), an essential constituent of the respiratory chain complex. We have shown that Atx inhibits CCOX activity when incubated with isolated mitochondria, an effect observed also on rat brain tissue sections. Interestingly, the effect does not seem to solely depend on Atx's phospholipase activity, as demonstrated with the enzymatically inactive mutant of the toxin. The results raised the question whether GIIA also interacts with this receptor and affects its enzymatic activity. We have, therefore, expressed and isolated the recombinant rat GIIA and its enzymatically inactive mutant and fully characterized them. Using heterologous competition assay, we demonstrated that the mammalian GIIA binds to CCOX-II with a 100-fold lower affinity than Atx. Homologous competition assay using radiolabeled GIIA, however, revealed that the endogenous sPLA2 primarily targeted another binding protein in mitochondria, which has an apparent mass of around 20 kDa (R20). Interestingly, GIIA and its mutant also inhibited CCOX activity on isolated mitochondria, suggesting that R20, a newly detected receptor, might be a subunit of CCOX and indicating a possible regulatory role of the endogenous GIIA in mitochondria. Taken together, our results suggest the explanation of the mechanism by which β-ntxs hinder the production of ATP in the poisoned nerve ending and open an important direction of study to advance the understanding of the involvement of the mammalian GIIA in mitochondrial function and dysfunction.

The seminar will be held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

23. 1. 2020 Anja Pavlin (BF): The bacteriophage GIL01 take-over mechanisms of the host`s SOS response

Abstract

The GIL01 bacteriophage, is a tectiviral temperate phage, which infects the insect pathogen Bacillus thuringiensis. The lytic cycle of GIL01 is induced as part of the cellular SOS response with which bacteria identify and respond to DNA damage. Unlike most temperate phages, GIL01 lysogeny is not established by a dedicated phage repressor, but rather by the host’s regulator of the SOS response, LexA. However, LexA is unable to maintain lysogeny, unless the small phage-encoded protein gp7 is also present. Gp7 directly interacts with LexA to enhance its DNA binding to phage promoter. We obtained the crystal structure of the 50-amino acid gp7 protein and according to SAXS data, we generated a structural model of gp7 in complex with LexA, which illustrates that gp7 positions LexA in a DNA bound conformation. By applying surface plasmon resonance approach we also recently discovered, that gp7 homologs identified in other tectiviruses, that infect a diverse range of bacteria, exhibit similar mode of action as gp7. Our recent results furthermore show, that a small protein non-homologous to gp7 in an important human pathogen, analogously to gp7, interacts with its cognate DNA damage response repressor to modulate the SOS response. Thus this results lead us to think that this kind of mechanism is widely spread to enable the SOS control beyond the LexA/RecA regulation. To determine how GIL01 establishes the lytic cycle, we examined the regulatory mechanisms at the lytic promoter. We show that lytic promoter is also repressed by LexA/gp7 complex and that the second phage-borne small protein, gp6, is the key activator of the lytic cycle. Surprisingly, gp6 is homologous to LexA itself and, thus, is a rare example of a LexA homologue directly activating transcription. We propose that the interplay between these two LexA family members, with opposing functions, ensures the timely expression of GIL01 phage late genes.

Caveney NA, Pavlin A, Caballero G, Bahun M, Hodnik V, Castro L, Fornelos N, Butala M, Strynadka NCJ. Structural Insights into Bacteriophage GIL01 gp7 Inhibition of Host LexA Repressor. Structure. 2019.

Fornelos N, Browning DF, Pavlin A, Podlesek Z, Hodnik V, Salas M, Butala M. Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue. Nucleic Acids Res. 2018.

The seminar was held at 14.30 in the lecture room B3 at the Biotechnical Faculty (BF) of the University of Ljubljana.

19. 12. 2019 Eva Jarc Jovičić (JSI): Are inflammatory lipid mediators derived from membranes or neutral lipids?

Abstract

Eicosanoids are potent lipid mediators derived from arachidonic acid, an ω-6 fatty acid, that regulate inflammation, immunity and tumourigenesis. They are released in large pools upon cell activation and are necessary for the proper execution of the inflammatory response. In contrast, the relatively novel and still expanding group of specialized pro-resolving mediators (SPMs) comprises derivatives of the ω-3 eicosapentaenoic and docosahexaenoic acids. SPMs predominantly display anti-inflammatory effects and are essential for the proper resolution of inflammation. The production of lipid mediators depends on the availability of their fatty acid (FA) precursors and the activities of numerous proteins and enzymes that regulate FA trafficking between different cellular pools of lipids. Traditionally, the hydrolysis of glycerophospholipids in cellular membranes by phospholipase A2 enzymes has been considered the main source and stimulus for eicosanoid synthesis. However, new evidence suggests that FAs for lipid mediator production may be derived from various cellular and extracellular lipid sources and mechanisms, including lysosomes and cytosolic lipid droplets. Lipid droplets have been recently recognized as organelles with pleotropic functions in cell metabolism and signalling. In the seminar, we will present novel mechanisms that link lipid droplets with lipid mediator production and challenge the dogmatic view of phospholipase-driven lipid mediator synthesis.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

17. 10. 2019 Veno Kononenko (BF): Potential of 3-alkylpyridinium salt-nanoparticle (APS-NP) cotreatment for cancer therapy

Abstract

Lung cancer is still the leading cause of cancer related death and smoking is the major cause of it. Nicotine is responsible for smoking addiction, but it is also known it works as a promoter for tumor growth. The mechanism for cancer development is very complex and not well understood, but it is known that nicotine activates nicotinic acetylcholine receptors (nAChR) and this triggers different signaling pathways that lead to uncontrolled cell proliferation, prevention of apoptosis and promotion of angiogenesis. By using antagonists of nAChR, we may cause the opposite effects, increasing the apoptosis of cancerous cells and decreasing their proliferation rate. Alkylpyridinium compounds (APS), which were isolated from marine sponge Reniera sarai, act as antagonist of α7 nAChR (subtype of nAChR that is overexpressed in lung cancer cells), but they can also form pores in the membranes and act cytotoxic. Due to these properties, different APS have a potential to be used in the cancer therapy. In our study we are using different APS analogues (APS 3Cl and APS 7Cl), which were obtained by organic synthesis. As a test system we are using human lung adenocarcinoma cells A549. We compared cytotoxicity of APS 7Cl with cytotoxic effects of nanoparticle-APS co-treatment. For the co-treatment we used different solid NPs (TiO2, SPIONs, SiO2, Au), but we were not able to provoke higher cytotoxicity to A549 cells. For that reason we started with the production of porous gelatin NPs that can be synthesized in a way that enables integration of APS 7Cl into their structure. Gelatin NPs are non-toxic and biodegradable, what makes them appropriate candidate for drug carriers and other medical applications. For the gelatin NP synthesis, we used nanoprecipitation method, by utilizing different surfactants (Tween-20, N-lauroyl sarcosinate, Triton X-100, and Poloxamer 407) as an emulsifying agents. The synthesized gelatin particles were observed by SEM. For further experiments with A549 cells, we chose gelatin NPs that were synthesized using Poloxamer 407. By incorporation of APS 7Cl into the gelatin NPs, we were able to achieve moderate cytotoxicity to A549 cells. We are witnessing several issues that will be addressed in the future studies: How to achieve higher incorporation/adsorption of APS into/onto gelatin NPs that will lead to higher cytotoxicity to cancerous cells? How to determine the amount of APS that is incorporated into gelatin NPs during the NP synthesis? What is the mechanism that leads to cytotoxicity of APS loaded gelatin NPs? How gelatin NPs affect non-cancerous cells? We will also test the performance of some other types of APS (and maybe some other molecules) for the selective cytotoxicity to cancerous cells.

The seminar was held at 14.30 in the lecture room B7 at the National Institute of Biology.

19. 9. 2019 Mojca Ogrizović (JSI): Intra-organelle communication through Pex11 in yeast Saccharomyces cerevisiae

Abstract

The degree of chromatin compaction is regulated through histone acetylation. Lightly packed chromatin is important for active gene transcription, whereas tightly packed chromatin is associated with gene silencing. Pex11 is a peroxisomal membrane protein involved in biogenesis of peroxisomes, the only organelles where in yeast β-oxidation of fatty acids takes place. In this metabolic process acetyl-CoA is produced, which is a crucial molecule in central carbon metabolism, and at the same time the substrate for histone acetylation. Our group performed a transcriptomic study where we discovered that deletion of PEX11 gene in yeast affects the expression of genes encoding glycolytic enzymes. We found that Pex11 forms a tether between peroxisomes and mitochondria, enabling close proximity of these organelles (Mattiazzi Ušaj et al., 2015). We hypothesized that by doing this Pex11 affects transport routes of acetyl-CoA from peroxisomes to mitochondria. We therefore tested whether Pex11 affects histone acetylation. Using western blot and immunodetection we show that in the pex11Δ deletion mutant cells acetylation of H4 subunit at positions K5 and K8 is severely reduced compared to the wild-type strain cells. Furthermore, we show that the deletion of PEX11 gene reduces histone acetylation to a lower level than mutations causing complete loss of peroxisomes, inactivation of β-oxidation of fatty acids or inactivation of respiration in mitochondria. Our experiments elucidated intra-organelle communication, not only through physical interaction, but also through connecting organelle-specific metabolic pathways at the cellular level.

Mattiazzi Ušaj M., Brložnik M., Kaferle P., Žitnik M., Wolinski H., Leitner F., Kohlwein S. D., Zupan B., Petrovič U. 2015. Genome-wide localization study of yeast Pex11 identifies peroxisome- mitochondria interactions through the ERMES complex. Journal of Molecular Biology, 427, 11: 2072-2087

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

16. 5. 2019 Maja Grundner (BF): DNA sampling II- method for isolation and characterisation of DNA-protein complexes

Abstract

Treatment of many bacterial infections has become challenging as the antibiotic resistance is now days responsible for thousands of deaths around the world. Bacteria respond to changing environment by tuning their transcriptional program and by doing so they can overcome the antibiotic treatment. Therefore in search for new antimicrobials, it is very crucial to understand the mechanisms, involved in development of antibiotic resistance.

Our aim is to improve a method »DNA sampling« published in 2009 by Butala et al. for identifying transcriptional factors at selected promoters and intragenic regions in order to gain knowledge on how toxins and other virulence traits are controlled.

For this method we engineered plasmids that function in couples. First plasmid carries the molecular tools needed to execute the method and the second plasmid the target DNA fragment to be sampled for proteins. The molecular tool plasmid carries under the arabinose inducible promoter: strep-tagged, fluorescently labelled LacI repressor, yeast nuclease I-Scel and lambda Gam protein. The target DNA fragment carries the two LacI sites upstream of the DNA fragment flanked by the I-SceI sites.

After arabinose addition, induction of the molecular program, I-SceI excises the target DNA fragment which is afterwards affinity isolated via the LacI-strep-tagged protein. By optimizing the isolation we managed to isolate the DNA fragment but weren’t successful in detecting specific proteins attached on the DNA due to unstable protein-DNA interactions. Thus, we are enhancing the method by directing the selected nucleoprotein complexes inside the 100 nm proteinaceous particles, named micrompartments. This will enable us to detect fluorescently labeled transcription factors that interact with the target DNA directly in the cell.

The seminar was held at 14.30 in the lecture room B4 at the Biotechnical Faculty (BF) of the University of Ljubljana.

18. 4. 2019 Gašper Žun (FKKT): New prospects in polygenic traits analysis

Abstract

Polygenic traits dissection is an ultimate goal of genetics, as phenotype is laboriously tried to be explained on molecular level. Since complex quantitative traits are of great importance, for instance in healthcare and bio-industry, their dependence on genetic elements, i.e. DNA, has to be precisely evaluated.

Yeast Saccharomyces cerevisiae offers a unique approach to dissect a desired polygenic trait: two distinct parental strains are crossed and such a diploid zygote is sporulated. As a consequence of independent segregation and crossing-over, a pool of genetically different segregants is collected.

With further phenotyping one can distinguish between different levels of quantitative trait expression. To demonstrate that, we manage to discriminate between neutral lipids reach and poor segregants with fluorescent dye staining. Similarly, salt-tolerance experiment is in progress.

When segregants with desired level in polygenic trait are discriminated, sequencing and comparison of their genomes provides quantitative trait elements. To clarify, such statistical data suggest which parts of genome are most likely to be responsible for desired quantity in polygenic trait. In particular, when crossing lipid rich and poor parental strain, PIG1 gene was suspected to be a quantitative trait gene for neutral lipids accumulation.

Since higher resolution into quantitative trait elements is preferable, we applied multiple steps of crossing. The segregant with the most desirable features in each stage was selected and mated with one of two starting parents. Further selection would dissolve and narrow responsible genetic loci within parental strain genome.

Such statistical prediction, being wide or narrow, has to be biologically evaluated. We applied CRISPR technique to successfully swap quantitative trait genes. What is more, with complementary application of two distinctive endonucleases, Cas9 and Cpf1, we are available to target almost every position within yeast’s genome. Additionally, we observed no predicted inserts and deletions at the recognition site of the CRISPR system. Under those evidences we can conclude that single nucleotide change within whole yeast genome can be changed, most likely without additional alterations.

Conclusively, we found and biologically proved quantitative trait gene for neutral lipids accumulation. For such a purpose we developed and evaluated CRISPR-Cas9 and -Cpf1 approach for precise gene and single nucleotide swap within a genome. Such an approach will be of great value in further polygenic traits dissection.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

21. 3. 2019 Anastasija Panevska (BF): New insights into the unique binding of ostreolysin A with lipid membranes

Abstract is not available.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

14. 2. 2019 Adrijan Ivanušec (JSI): Characterization of interaction between sPLA₂s and mitochondria

Abstract

Secretory phospholipase A₂ group IIA (GIIA sPLA₂) is a structural homologue of ammodytoxin (Atx), a β-neurotoxic GIIA sPLA₂ from the snake venom, and it plays both physiological and pathophysiological roles in mammalian brain. Physiologically, this enzyme seems to play a part in the neurotransmission, neuritogenesis and mitochondrial homeostasis, while pathologically, it is involved in the neurodegenerative (e.g., Alzheimer’s disease) and cerebrovascular diseases (e.g., stroke). In the case of Alzheimer's disease, GIIA sPLA₂'s expression and activity are elevated in the affected tissue, accompanied by neurodegeneration with characteristic dysfunction of neuronal mitochondria. While the molecular mechanism of action of GIIA sPLA₂ is not known, the effects of GIIA and β-neurotoxins (β-ntxs) on mitochondria are similar, therefore a description of the mode by which β-ntxs encounter and affect neuronal mitochondria on the molecular level would be expected to advance the study of the role of endogenous GIIA sPLA₂ in mentioned diseases, ultimately leading to new diagnostic and therapeutic solutions. In that regard, high affinity membrane receptor for Atx was recently identified as the subunit II of cytochrome c oxidase (CCOX-II), an essential constituent of the respiratory chain complex. It was shown that the toxin inhibits CCOX activity when incubated with isolated mitochondria, an effect which may not solely depend on Atx activity. To further investigate the role of enzymatic activity of Atx in its interaction with mitochondrion, we have labelled Atx(D49S), an enzymatically inactive mutant of Atx, with 5 nm NHS-activated gold nanoparticles and are studying its internalization into PC12 cells and translocation into mitochondria by the means of transmission electron microscopy. While the interaction with CCOX-II seems to be exclusive for Atx among β-ntxs, the question arises whether the endogenous GIIA sPLA₂ also interacts with this receptor and affects its enzymatic activity. We are planning to tackle this question with heterologous competition binding assays using ¹²⁵I-AtxC and CCOX activity interference test. We have, therefore, expressed and isolated a recombinant rat GIIA sPLA₂ and fully characterized it in terms of its molecular mass, N-terminal amino acid sequence and enzymatic activity. In the future we are also planning to investigate whether Atx and GIIA sPLA₂translocate into neuronal cells by exploiting the same path of translocation.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

24. 1. 2019 Matej Skočaj (BF): Aegerolysin-like proteins as new theranostic tools in periodontal disease and prosthetic rehabilitation

Abstract

Periodontal diseases (PD) represents a group of chronic oral inflammatory infections initiated by oral periodontal pathogens which can be found in bacterial plaques, attached to the tooth surface. The disease begins as chronic local inflammation of the gingival tissue (gingivitis) and can progress to periodontitis that is characterized by degradation of the periodontal ligament and alveolar bone, which eventually leads to formation of periodontal pockets and ultimately results in tooth loss. In adults, periodontitis is a major cause of tooth loss in both developed and developing countries. In adults over 65 years PD were confirmed by as many as 70% of individuals, and due to general aging of the population and longer retention of natural dentition this number is expected to increase even more.

To date, reliable molecular tools for defining susceptibility to PD, its early detection and monitoring of treatment success are not known. Diagnosis of PD is made strictly on the basis of the evaluation of clinical signs and symptoms of inflammation. The diagnosis is supported by evidence from radiographs, which provide additional diagnostic tool and can demonstrate the presence of marginal bone loss. Therefore, the problem in early diagnostics of periodontitis is that the diagnosis is based upon an assessment of the already apparent tissue breakdown. Up to date, none of the candidate molecules has shown the potential to act as an accurate, precise and reproducible indicator of the destructive processes linked to PD.

One of the potential molecules that can be considered as a biomarker of periodontal disease is a specific sphingolipid molecule, ceramide phosphoethanolamine (CPE), as well as its dihydrogenated form, dihydroceramide phosphoethanolamine (DCPE). CPE and DCPE are the major sphingolipids in cell membranes of invertebrates, parasites and some Gram-negative bacteria. CPE and DCPE are characteristic lipids for prokaryotic phylum Bacteroidetes. Human oral cavity is inhabited by multiple bacterial species proficient in CPE and DCPE biosynthesis including Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia, which belong to the phylum Bacteroidetes. They are highly abundant in dental plaque samples of chronic periodontitis patients, and represent the main causative bacterial agents of PD.

Recently it was revealed that aegerolysins can be utilised as efficient molecular tools for detecting and visualizing CPE. Aegerolysins PlyA2, OlyA and erylysin A (EryA) strongly associate with CPE/Chol- containing membranes, and this association is 1000-fold higher compared to SM/Chol membranes. In particular, EryA binds exclusively to CPE and dose not bind to SM. Further, aegerolysins show pore- forming activity, when combined with pleurotolysin B (PlyB), a 59-kDa protein partner with a membrane-attack-complex/perforin (MACPF).

Our results show that aegerolysins act as bi-component pore-forming complexes on artificial and biological cell membranes containing CPE. Therefore, we propose the use of (i) CPE-sensing aegerolysin EryA for detection of PD and (ii) the combination of CPE-sensing EryA and MACPF domain containing PlyB for specific eradication of CPE-containing periodontal bacteria. It is known that the framework of the commensal oral microbiota consists primarily of Gram-positive bacteria. Since Gram-positive bacteria do not contain CPE/DCPE, we assume the specificity of antimicrobial activity on Gram-negative CPE-containing bacteria that are responsible for the aetiology of periodontitis, without harmful effects on the commensal microbiota and to the host.

The seminar was held at 14.30 in the lecture room B3 at the Biotechnical Faculty (BF) of the University of Ljubljana.

20. 12. 2018 Cene Gostinčar (BF): Ekstremna okolja - ekstremni genomi?

Abstract

Zaradi hitrega razvoja tehnologije določanje zaporedij celotnih genomov že lep čas ni več (izključno) področje velikih raziskovalnih konzorcijev in vrtoglavih proračunov. Genomika se je s tem premaknila onkraj meja dobro preučenih modelnih organizmov in začela odkrivati izjemno raznolikost živega sveta.
V zadnjih dveh desetletjih se je izkazalo, da nekaterih od najbolj ekstremnih okolij na planetu ne naseljujejo le arheje in bakterije, temveč tudi glive. Te preživijo tako nizke temperature in pomanjkanje hranil v arktičnih ledenikih, kot tudi visoke koncentracije soli v obmorskih solinah in slanih jezerih. Razumevanje prilagoditev na ekstremne razmere je v osnovi vprašanje temeljne znanosti, ima pa tudi pomemben potencial v različnih biotehnoloških procesih. Ker gre za organizme, s katerimi v laboratoriju ni enostavno rokovati, je bilo njihovo preučevanje – tudi zaradi nepoznavanja genomskih zaporedij – od vsega začetka relativno zahtevno in počasno. Da bi si delo olajšali, smo po letih molekularnih raziskav pred nekaj leti določili prve genome halofilnih in ekstremo halotolerantnih gliv, kasneje pa smo se posvetili še nekoliko manj ekstremofilnim, a zato toliko bolj prilagodljivim generalističnim glivam.
Preučevanje genomskih zaporedij, s katerim se v skupini ukvarjamo vse bolj intenzivno, nam je odgovorilo na marsikakšno vprašanje, še več pa nam jih je zastavilo. Predavanje se bo dotaknilo prvih in drugih vprašanj, predvsem pa bo poskušalo na nazoren način prikazati izzive in priložnosti, ki jih nudi genomika v laboratorijih manj pogostih, morda nekoliko eksotičnih, vsekakor pa nadvse zanimivih organizmov.

The seminar was held at 14.30 in the lecture room B5 at the Biotechnical Faculty (BF) of the University of Ljubljana.

19. and 21. 11. 2018 A 2-day Mini-Symposium of the Programme Group (JSI & BF)

On 19 and 21 November we organized a traditional Mini-symposium of our Programme Group. On this occasion members of the group presented their research results and projects. As well, some guests, representatives of the partner groups, presented their projects and joint activities. The event was of an informal and working character to maximally stimulate a critical discussion about the presented results and ideas, aiming to reveal novel collaboration possibilities. The detailed programme of the meeting can be found here.

18. 10. 2018 Valerija Vezočnik (BF): Kinetically stable triglyceride-based nanodroplets

Abstract

Understanding of the interactions between proteins and natural and artificially prepared lipid membrane surfaces and embedded nonpolar cores is important in studies of physiological processes and their pathologies and is applicable to nanotechnologies. In particular, rapidly growing interest in cellular droplets defines the need for simplified biomimetic lipid model systems to overcome in vivo complexity and variability. 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. Lipid composition, ζ-potential, gyration and hydrodynamic radius, shape, and temporal stability of the lipid nanodroplets were characterized and compared to extruded SM/Chol large unilamellar vesicles. Lipid nanodroplets and large unilamellar vesicles 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 (ζ-potential, orientational order of interfacial water molecules) and binding of these proteins to the nanodroplet SM/Chol monolayers were similar to those for the SM/Chol bilayers of the large unilamellar vesicles 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.

The seminar was held at 14.30 in the lecture room B3 at the Biotechnical Faculty (BF) of the University of Ljubljana.

27. 9. 2018 Eva Jarc (JSI): Between death and survival: lipid droplets protect cancer cells against nutrient stress

Abstract

Cancer cells have the ability to thrive under continuous nutrient and oxidative stress. Some of the most aggressive tumours are masters in balancing nutrient acquisition and consumption with the energy demand required for cell survival. During hypoxia and glucose deprivation, cancer cells shift from their dependence on glucose and rely on mitochondrial fatty acid (FA) oxidation for energy production. Cancer cells acquire FAs either through FA uptake from the microenvironment or through de novo FA synthesis, while excess free FAs are stored in the form of triacylglycerols (TAGs) in cytosolic lipid droplets (LDs). In the past decade, LDs have been recognized as dynamic organelles providing lipids for energy production, membrane synthesis and lipid-mediated signalling. Recent studies have shown that LD biogenesis is increased in cells exposed to various types of stress, including complete nutrient deprivation, hypoxia or nutrient excess, suggesting that LDs are important for the cellular stress response. Here we examined the role of LDs in the protection against nutrient and lipotoxic stress in aggressive breast cancer cells. By modulating LD formation and breakdown, we show that LDs protect breast cancer cells during starvation-induced nutrient stress and reduce the lipotoxicity of exogenously added unsaturated FAs. Thus, the balance between LD synthesis and breakdown is a potential metabolic vulnerability of cancer cells that can be targeted in cancer therapy.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

14. 6. 2018 Jernej Šribar (JSI): Ammodytoxin-binding proteins and their potential role in the process of neurotoxicity

Abstract

Ammodytoxins (Atx), presynaptically neurotoxic secreted phosholipases A₂ (sPLA₂s) are one of the major components of the Vipera ammodytes ammodytes venom. Results of numerous studies have revealed that the presynaptically neurotoxic mechanism of action of these and that of similar neurotoxic sPLA₂s is a result of both their enzymatic activity and binding to specific receptors in the nerve terminals of motor neurons. At the neuro-muscular junction, they arrest the secretion of acetylcholine into the synaptic cleft and thus block the communication with the neighbouring muscle cell, causing the death of the victim due to respiratory muscles paralysis. With the discovery and characterization of several Atx-binding proteins, and the study of the role of Atx enzymatic activity, we have significantly contributed to the explaining of the molecular mechanism of these toxins and their localization within the cells. Furthermore, our findings will provide an insight into the understanding of the pathophysiological roles of orthologous mammalian sPLA2s. An overview of the current comprehension of the neurotoxic action of Atx will be presented.

The seminar was held at 15.30 in the lecture room B3 at the Biotechnical Faculty (BF) of the University of Ljubljana.

14. 6. 2018 Maruša Novak (BF): Fungal and bacterial aegerolysins: binding to artificial and biological membranes

Abstract

Proteins of the aegerolysin family (Pfam06355) are relatively widely distributed in bacteria and fungi, but also appear in plants and insects and therefore span many kingdoms of life. These small, ~13 to 20 kDa, β-structured proteins are known to bind to different lipids, soluble lipid derivatives and membrane domains. Moreover, aegerolysins can play a direct role in pore formation or help other pore forming proteins (e.g. membrane attack complex/perforin (MACPF) domain-containing proteins) to assemble in to bi-component transmembrane pores. Due to all this, aegerolysins have biotechnological potential as markers of membrane lipids and also as potential anti-cancer drugs or insecticides.
Recently, we have confirmed that the common binding target for aegerolysins RahU from the bacterium Pseudomonas aeruginosa, NigA2 from the filamentous fungus Aspergillus niger and OlyA from the edible mushroom Pleurotus ostreatus is a sphingolipid, ceramide phosphoethanolamine (CPE). This lipid is abundant in the cell membranes of invertebrates (especially insects) and protozoa, whereas cell membranes of vertebrates contain only trace amounts of it. Binding characteristics of RahU, NigA2 and OlyA aegerolysins to lipid vesicles containing CPE and lipid vesicles composed of lipids derived from the Sf9 cells (cell line from the insect Spodoptera frugiperda) will be presented in the seminar along with the nuclear magnetic resonance and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of non-polar lipids of Sf9 cells.

The seminar was held at 14.30 in the lecture room B3 at the Biotechnical Faculty (BF) of the University of Ljubljana.

17. 5. 2018 Valerija Vezočnik (BF): Nanoemulsions of lipid droplets as a model lipid system

Abstract

In this study, we introduce nanoemulsion-based lipid droplets and their use in studying protein-lipid interactions as a new biomimetic lipid model, complementary to the lipid bilayers, and monolayers formed at the water/air interfaces (Langmuir monolayers). Specifically, stable lipid nanodroplets, i. e. nanoemulsions of lipid droplets (LDs) composed of trioleoylglycerol core covered by a monolayer of sphingomyelin (SM) and cholesterol (Chol), have been prepared by our recently developed combined reverse-phase evaporation/ultrasonication method. Prepared LDs were examined for lipid composition, polydispersity, stability, and interaction with sensor chips by using several (bio)chemical and (bio)physical techniques. To our knowledge, this is the first in-depth characterization of nanoemulsion covered by a SM/Chol monolayer. In parallel, Langmuir monolayers and large unilamellar vesicles (LUVs) composed of SM and Chol have been prepared. All three lipid membrane models with SM/Chol molar ratios of 1/1 and 4/1 were used to study interaction with a Chol-binding protein, perfringolysin O (PFO). We found that PFO binds comparably well to monolayers and bilayers containing Chol above 20 mole-%. Moreover, PFO binding to LDs appeared similar to that observed with LUVs and Langmuir monolayers. Therefore, we propose that LDs enable to study pore formation as well. In conclusion, our findings suggest that artificial LDs are a suitable biomimetic lipid system for studying protein-lipid interactions complementary to the lipid vesicles and Langmuir monolayers.

The seminar was held at 14.30 in the lecture room B3 at the Biotechnical Faculty (BF) of the University of Ljubljana.

18. 4. 2018 Adrijan Ivanušec (JSI): Characterization of the interaction between sPLA2s and mitochondria

Abstract

β-neurotoxic snake venom secreted phospholipases A2 (sPLA2s) block neuro-muscular transmission by poisoning nerve terminals of a motoneuron. Effects of these toxins include damage to neuronal mitochondria, which is very similar to that induced by a structurally homologous endogenous group IIA sPLA2 (GIIA sPLA2) in certain pathological conditions. The molecular mechanism of action of β-neurotoxins is not yet fully understood. A high affinity membrane receptor for ammodytoxin A (AtxA), a β-neurotoxic sPLA2 from the venom of the nose-horned viper (Vipera ammodytes ammodytes), was discovered in neuronal mitochondria and recently identified as the subunit II of the cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. It was shown that the toxin inhibits CCOX activity when incubated with isolated mitochondria, an effect which may not solely depend on Atx activity. The exact mechanism of this inhibition and how the toxin translocates into mitochondria, however, remains to be investigated. To study these processes we have prepared several molecular tools. We have expressed and isolated a recombinant protein AtxA(D49S), an enzymatically inactive mutant of Atx, and performed its thorough biochemical characterization. We determined its molecular mass, N-terminal amino acid sequence, enzymatic activity and binding to calmodulin, an intracellular Atx-binding protein. By the means of heterologous competition with ¹²⁵I-AtxC binding we have confirmed that not only wild type Atx, but also AtxA(D49S) bind to CCOX-II. To investigate the translocation of the enzymatically inactive mutant into mitochondria of PC12 cells, a neuron-like model cell line, we have labelled AtxA(D49S) with 5 nm NHS-activated gold nanoparticles and will study its localization by the means of transmission electron microscopy. The question arises whether endogenous GIIA sPLA2, a structural homolog of AtxA, also interacts with this mitochondrial protein and affects its enzymatic activity. In the future, we will try to answer this question by producing recombinant rat GIIA sPLA2 and use it for affinity labelling of CCOX and CCOX activity interference test on isolated mitochondria.

The seminar was held at 8.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

22. 3. 2018 Zorica Latinović (JSI): Expression of snake venim proteins in a mammalian cell line

Abstract

Snake venom is a source of molecules interfering with different parts of the haemostatic system. One of the most venomous snake in Europe, which venom causes local effects and systemic manifestations associated with bleeding and coagulopathies is Vipera ammodytes ammodytes (Vaa). The effects of this venom on haemostasis can be assigned to disintegrins (Dis), C-type lectin-like proteins (snaclec), secretory phospholipases A2 (sPLA2), metalloproteases (MP) and serine proteases (SP). In the Vaa venom, we detected a glycoprotein, which significantly prolongs the blood clotting time. It is a monomeric N-glycosylated protein of 35 kDa, structurally similar to SPs, but devoid of enzymatic activity thus named Vaa SP homolog 1 (VaaSPH-1). The anticoagulant effect of VaaSPH-1 is mostly due to its binding to coagulation factor VIIIa. As a potent non-enzymatic inhibitor of blood coagulation pathway, VaaSPH-1 is the unique and very promising therapeutic candidate for the treatment of venous thromboembolism (VTE). However, for further in vivo studies of its anticoagulant potential, i.e. for experiments on animals, we need to have sufficient quantity of VaaSPH-1. The amount isolated from Vaa venom is very low (VaaSPH-1 represents only 0.002% of the whole venom proteins), which can additionally contain traces of other snake venom proteins, with possible effect on blood coagulation system. Furthermore, due to the many disulphide bridges in the VaaSPH-1 structure and N-glycosylation pattern on the protein, the production in bacteria and yeast is not suitable. Hence, there is an increasing demand for production of VaaSPH-1 in animal cell culture. Mammalian HEK293 FreeStyle expression system enables the high yield production of post-translationally modified recombinant proteins through suspension-growing, serum-free adapted cell lines. For expression in HEK293 cell line we used expression cloning vector pcNA3.1 in which we inserted optimized DNA sequence of VaaSPH-1, with Kozak sequence, signal peptide, human codons, His-tag and TEV cleavage site for removal of His-tag. With linear polymer polyethylenimine (PEI) we performed direct and indirect transfections, with more promising result gained with the former one, and further optimized transfection protocol with addition of valproic acid (VAP). The cells successfully expressed VaaSPH-1 into the media and after the purification with nickel-affinity chromatography we tested it for its anticoagulant activity. To this end, we were able to express active snake venom protein in the mammalian HEK293 FreeStyle cell line. The recombinant protein has the appropriate molecular mass and defined anticoagulant activity as does the native protein from Vaa snake venom.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

15. 2. 2018 Vid Leban, Mojca Dobaja Borak (University Medical Centre Ljubljana): Influence of C-type lectin-like proteins (snaclecs) in the Vipera ammodytes ammodytes venom on haemostasis

Abstract

C-type lectin-like proteins (snaclecs) are the heterodimeric proteins found in the venom of many species of the Viperidae family. They are interfering with haemostasis by acting on platelet receptors. Nine nucleotide sequences of snaclec monomers were found in a Vipera ammodytes ammodytes (Vaa) venom gland cDNA library. Based on preliminary data from in vitro studies, we predict their binding to the GPIb receptors, thereby preventing interaction with the von Willebrand factor and inhibiting the process of platelet adhesion to subendothelial collagen in vivo. They also induce thrombocytopenia in human, which is predominant but yet not fully understood haemotoxic effect of Vaa venom. Our research is focused on the mechanism of thrombocytopenia induced by snaclecs. We will present our first results of the isolation of Vaa snaclecs and the clinical value of thrombelastometry in the treatment of snake-bitten patients.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

25. 1. 2018 Sabina Ott (JSI): Phylogenomic analysis of RNA viruses in invertebrates

Abstract

RNA viruses are the largest group of viruses representing 54 out of 110 viral families. Among them are some of the most notorious human pathogens. RNA viruses have been grouped into 25 viral clades: Astro, Birna, Hepe-Virga, Hypo, Luteo-Sobemo, Narna-Levi, Bunya-Arena, Mono-Chu, Orthomyxo, Nido, Partiti-Picobirna, Permutotetra, Picorna-Calici, Reo, Tombus-Noda, Toti-Chryso, Weivirus, Yanvirus, Zhaovirus, Yuevirus, Qinvirus, Cysto, Poty, Flavi and Ophio (Shi et al., 2016).
Invertebrates possess numerous divergent RNA viruses. Many insect lineages (Diptera, Hemiptera, Polyneoptera, Odonata and Thysanoptera) and nematodes are vectors of plant and animal viruses. Invertebrates might represent the center of RNA viral biodiversity and evolution. Shi et al. (2016) has done a large-scale analysis of RNA viruses in Lophotrochozoa, nematodes and arthropods. However, some important invertebrate groups have not been thorougly analysed for novel RNA viruses, namely 1) Platyhelminthes, 2) insect lineages Hymenoptera and Lepidoptera, 3) Crustacea – mainly Amphipoda and Copepoda, 4) the oldest lineages of Metazoa - Cnidaria, Porifera, Ctenophora, and 5) the oldest hexapod lineages – Collembola, Diplura, Zygentoma and Monocondylia. Our aim was to obtain an in depth insight into the RNA viromes of these taxa.
We explored the diversity and distribution of RNA viruses in oldest hexapod, oldest metazoan, crustacean, lepidopteran, hymenopteran and lophotrochozoan lineages by homology searching of the transcriptomic (TSA) databases. Genomic databases (WGS, nr, Refseq_genomic, Refseq_representative_genomes) of these lineages were analysed for novel RNA endogenized viral elements (EVEs). In a phylogenetic analysis, we have placed novel RNA viruses in the context of RNA viral families and clades. A comparative analysis of their genomes has been done with respect to well established and floating viral genera. We have also analysed the contribution of RNA viral clades to the RNA virome in analysed invertebrate groups.
815 novel RNA viruses belonging to 22 out of 25 RNA viral clades have been found in our study. The following RNA viral clades were found in all analysed invertebrate groups: Hepe-Virga, Narna-Levi, Mono-Chu, Orthomyxo, Partiti-Picobirna, Picorna-Calici, Tombus-Noda and Toti-Chryso. The most abundant groups among ss(-)RNA, dsRNA and ss(+)RNA viruses are: Mono-Chu, Partiti-Picobirna and Picorna-Calici. A number of novel RNA viruses formed new groups inside the defined viral clades. The whole genomes for numerous oldest representatives of diverse RNA viral clades have been found. 12 RNA viral clades were found endogenized in the analysed genomes. Mono-Chu was the only clade found endogenized in all analysed invertebrate phyla. Our analyses have demonstrated that the analysed invertebrate groups have unique patterns of viral clades in the RNA virome and unique collections of divergent RNA viruses. To conclude, we have shown that invertebrates are the center of RNA viral diversity and evolution.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

21. 12. 2017 Vesna Hodnik (BF): Specificty of microbial cytolysins

Abstract

Rastlinski patogeni mikroorganizmi imajo pester nabor molekul za okužbo rastlin, ki jih uporabljajo za infekcijo in širjenje po listih in drugih tkivih. Pri bakterijah, glivah in oomicetah je že nekaj časa poznana družina proteinskih molekul, imenovana NLP. Ti proteini so udeleženi pri boleznih številnih kmetijsko pomembnih rastlin, kot so krompir, paradižnik, soja, itn. En od odgovorov rastlin na proteine NLP je odmiranje rastlinskega tkiva, do katerega pride pri dvokaličnicah, ne pa pri enokaličnicah, kot so koruza, pšenica ali riž. V raziskavi, ki je bila pravkar objavljena v prestižni reviji Science, smo razložili, katera molekula rastline je prva v stiku s proteinom NLP in zakaj so enokaličnice neobčutljive na proteine NLP. Rastlinske bolezni povzročajo velike ekonomske izgube na račun okužb rastlin, zato raziskava predstavlja pomemben korak k razumevanju toksične aktivnosti proteinov NLP in odpira možnosti za razvoj molekul, ki lahko preprečijo delovanje proteinov NLP.

The seminar was held at 14.30 in the lecture room B7 at the National Institute of Biology.

21. and 23. 11. 2017 A 2-day Mini-Symposium of the Programme Group (JSI & BF)

On 21 and 23 November we organized a traditional Mini-symposium of our Programme Group. On this occasion members of the group presented their research results and projects. As well, some guests, representatives of the partner groups, presented their projects and joint activities. The event was of an informal and working character to maximally stimulate a critical discussion about the presented results and ideas, aiming to reveal novel collaboration possibilities. The detailed programme of the meeting can be found here.

19. 10. 2017 Eva Jarc (JSI): Lipid droplets regulate fatty acid storage and release to protect cancer cells from nutrient and oxidative stress

Abstract

Under aerobic conditions, mammalian cells maintain metabolic homeostasis by oxidizing glucose through glycolysis and mitochondrial oxidative phosphorylation. During hypoxia and glucose deprivation, cells shift from their dependence on glucose and rely on mitochondrial fatty acid (FA) oxidation for energy production. Cells acquire FAs either through FA uptake or de novo FA synthesis and store excess free FAs in the form of triacylglycerols (TAGs) in cytosolic lipid droplets (LDs). Emerging evidence suggests that besides storing FAs, LDs also coordinate cellular FA metabolism and stress responses. Cancer cells fight against stress by reprogramming their lipid metabolism to ensure sufficient FA levels for tumour growth. Moreover, they are able to take up high amounts of exogenous FAs and store them within LDs. Elevated LD content has been observed in several cancer types and correlates with tumour aggressiveness and resistance to chemotherapy. Although cells depend on FAs for mitochondrial energy production, an excess of FAs leads to lipotoxicity and cell death. Thus, the balance between FA storage and use must be tightly regulated in all cells exposed to stress and should be particularly critical for tumour growth and metastasis. Here we examined the role of LDs in the protection against nutrient and lipotoxic stress in aggressive Ras-driven breast cancer cells. We also addressed the issue of how different unsaturated FAs affect the survival of breast cancer cells. By silencing the rate-limiting enzyme in LD breakdown, adipose triglyceride lipase (ATGL), by inhibiting LD formation and by modulating the unsaturation levels of triglycerides stored in LDs, we show that LDs protect sensitive ω-3 and ω-6 polyunsaturated FAs (PUFAs) from oxidation by storing them in the form of inert triglycerides, while concurrently providing FAs for mitochondrial energy production, redox homeostasis and cell survival. LDs thus emerge as antioxidant and pro-survival organelles that balance unsaturated FA trafficking with cell survival mechanisms to protect cancer cells from stress. Our results suggest that LD metabolism is a critical metabolic vulnerability and a viable target in the fight against aggressive breast cancer.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

28. 09. 2017 Anastasija Panevska (BF, UL): Protein complexes from the fungal genus Pleurotus, new bio-pesticides for controlling Colorado potato beetle and Western corn rootworm

Abstract is not available.

The seminar was held at 14.00 in the lecture room B3 at the Biotechnical Faculty (BF) of the University of Ljubljana.

15. 06. 2017 Anastasija Panevska (BF, UL): Interaction of aegerolysin proteins from the fungal genus Pleurotus with artificial lipid membranes

Abstract

Aegerolysin protein family (Pfam 06355, InterProIPS009413) comprises over 350 mostly β-structured, small (15-20 kDa) proteins from different bacterial and eukaryotic taxa. Their common feature is the ability to bind different lipids and lipid derivatives, as well as biological and artificial lipid membranes. Our recent findings show that almost all bacterial and fungal aegerolysins specifically recognize ceramide phosphoethanolamine (CPE), the major membrane sphingolipid of several invertebrate classes. Moreover, in the presence of partner protein with a membrane-attack-complex/perforin (MACPF) domain, fungal aegerolysins from the genus Pleurotus can permeabilize lipid membranes through the formation of bi-component transmembrane pore complexes.

We have successfully isolated and characterized three recombinant Pleurotus aegerolysins, namely ostreolysin A6, pleurotolysin A2 and erylysin A, and their MACPF-protein partner pleurotolysin B. Using surface plasmon resonance we monitored the binding of these aegerolysins (with or without pleurotolysin B) to large unilamellar vesicles containing CPE. Furthermore, we determined the interaction of some soluble lipid derivatives with the selected aegerolysins. Lytic activity of fungal aegerolysins with their partner MACPF-protein was assessed on calcein-loaded small unilamellar vesicles containing CPE.

We show that all the tested aegerolysins specifically interact with CPE-containing artificial membranes, and that this interaction is stabilized in the presence of pleurotolysin B. Moreover, these membranes/vesicles were permeabilised with aegerolysins and pleurotolysin B only if containing CPE.

Our findings give base to explore the possible lytic effects of fungal aegerolysins and their MACPF- protein partner on biological membranes with similar lipid composition, and their possible biotechnological use as tools for selective control of invertebrate pests containing CPE in their membranes.

The seminar was held at 15.00 in the lecture room B2 at the Biotechnical Faculty (BF) of the University of Ljubljana.

18. 05. 2017 Maruša Novak (BF, UL): Aegerolysins: binding properties and new targets

Abstract

Proteins of the aegerolysin family (Pfam06355) are relatively widely distributed in bacteria and fungi, but also appear in plants and insects and therefore span many kingdoms of life. This family comprises of more than 350 representatives, unevenly distributed among species. Aegerolysins are small, β-structured proteins (15-20 kDa). Although their biological role remains to be elucidated, they have one important characteristic in common: they can interact with different lipids, membrane domains or soluble lipid derivatives. Moreover, some aegerolysins have been shown to form pores in artificial or biological membranes in combination with a partnering protein, component B, resulting in lysis. Due to all this, aegerolysins have biotechnological potential as markers of membrane lipids and also as potential anti-cancer drugs or insecticides.
In our lab we developed protocols for production of recombinant aegerolysins deriving from different bacteria and fungi. Based on recent finding that aegerolysins can bind to ceramide phosphoethanolamine (CPE), sphingomyelin abundant in the membranes of invertebrates (especially insects and protozoa), we are now trying to better determine the binding characteristics of different aegerolysins to artificial membranes containing CPE. Some of our recent results will be presented. Since we observed that despite belonging to the same family, aegerolysins seem to differ in their binding characteristics to different lipids, membrane domains or soluble lipid derivatives, we are also trying to find new membrane targets to which aegerolysins may bind by isolating lipids from different organisms and testing binding of different aegerolysins to these lipids with TLC blots and use of multilamellar vesicles made of these lipids. This may help us to better determine their biological role/s.

The seminar was held at 14.30 in the lecture room B2 at the Biotechnical Faculty (BF) of the University of Ljubljana.

20. 04. 2017 Mojca Ogrizović (JSI): Regulation of neutral lipid accumulation and disadvantageous pleiotropic effect of MKT1^S288cin yeast Saccharomyces cerevisiae

Abstract

Most heritable traits are polygenic, including the majority of microbial biotechnologically important characteristics. Extreme quantitative trait loci mapping (X-QTL) is a method developed in yeast that enables detection of all genetic loci even with small effects on a given trait. X-QTL was applied to identify QTLs responsible for high neutral lipid content in a cross between two Saccharomyces cerevisiae strain, an inferior laboratory S288c strain and a superior industrial AWRI1631 strain. Within the discovered QTLs, three candidate causative genes were identified. With ongoing experiments, we are trying to prove the involvement of the candidate causative genes in regulation of neutral lipid accumulation. These experiments include all single, double and triple allele swaps from the superior to the inferior parent and all combinations of allele deletions in both backgrounds. The created mutants are now being analyzed for neutral lipid content. Through genotyping a significant number of F1 segregants for the candidate genes we already got good indicia that they are the casual genes. Moreover, with backcrossing the elite segregants of each generation to both parental strain until we reach a plateau and then sequencing the winning segregant of both lineages, we will reveal additional beneficial genes in both parents. Within the discovered QTLs, we found also a fourth gene, MKT1, which was found as a causal gene in several stress related quantitative traits, including drug resistance, ethanol tolerance, sporulation efficiency and high temperature growth. These studies were conducted between an inferior S288c isogenic laboratory strain and a superior non-related strain. MKT1^S288c has two mutations, D30G and K453R absent in other laboratory strains and natural isolates, where only the former has been proven to be involved in sporulation efficiency. Though plenty QTL studies have been done where MKT1²^88c seemed to be disadvantageous, they were based on either one cross, were focusing on one trait or lacked the follow-up experiments, failing to firmly prove the pleotropic defect of MKT1^S288c allele regardless of the strain background. Therefore, we will perform allele swaps of MKT1²^88c to several S288c non-related strains, including AWRI1631. Furthermore, since the above-mentioned mutations are being linked to the MKT1^S288cdefect we will exchange both S288c specific polymorphisms between the S288c and AWRI1631 strains. Then we will test the resulting mutants for quantitative phenotypes, elucidating the pleotropic effect of MKT1^S288c allele.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

23. 03. 2017 Zorica Latinović (JSI): Two serine proteases from the venom of the nose-horned viper (Vipera a. ammodytes) and their effects on the intrinsic blood coagulation pathway

Abstract

Two proteins with a prominent activity on hemostasis, a serine protease homologue, named VaaSPH-1, and a serine protease, SP-10, were purified from the venom of the most venomous European snake Vipera a. ammodytes (Vaa), in a two-step chromatographic procedure using gel filtration and ion-exchange chromatography. As estimated by SDS-PAGE analysis, both proteins are monomers of 34 kDa and N-glycosylated.
VaaSPH-1 significantly prolonged the activated partial thromboplastin time (aPTT) in human plasma, which indicates perturbations in the intrinsic pathway of blood coagulation. It is structurally similar to snake venom serine proteases. It possesses, however two mutations in its catalytic triad, which makes it enzymatically inactive. Detailed analysis of the mechanism of blood coagulation by VaaSPH-1 exposed that the molecule inhibits the activity of tenase and prothrombinase complexes, with IC₅₀ in nanomolar range. We demonstrated that the inhibition of the formation of the complexes is due to binding of VaaSPH-1 to blood coagulation factors constituting mentioned complexes. VaaSPH-1, which is a basic protein, binds also specifically to negatively charged phospholipids, phosphatidylinositol and phosphatidylserine, the latter being crucial for constitution of coagulation complexes on the blood cell membrane. Based on all experimental results we suggest that VaaSPH-1’s major anticoagulant action is due to binding to FIX/FIXa.
Contrary to VaaSPH-1, enzymatically active SP-10 acts procoagulantly. When added to the human plasma, it shortened the thrombin time (TT) and the re-calcification time in a dose-dependent manner. SP-10 specifically activated coagulation factors of prothrombinase complex - FX to FXa, expressing the FVIIa-like activity, and FV to FVa. It did not bind or activate FIX, FXI and FXII, and did not induce clotting of fibrinogen in the blood plasma.
Both snake venom proteins are unique hemostatically active snake venom proteins and therefore very interesting for further characterization towards the medical application as effectors of the intrinsic blood coagulation pathway: VaaSPH-1 as a potent non-enzymatic inhibitor and SP-10 as specific activator.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

22. 03. 2017 Toni Petan (JSI): Lipid droplets and the management of nutrient and oxidative stress in cancer

Abstract

The ability of cancer cells to survive stressful environmental conditions is crucial for tumour growth and metastasis. During stress, cancer cells adapt to alternative nutrient sources and modify their metabolism to match nutrient supply. Some of the most aggressive cancer cells, driven by the Ras oncogene, scavenge unsaturated fatty acids from their environment in order to cope with hypoxia and nutrient deprivation. The mechanisms of fatty acid uptake and the cellular metabolic adaptations that enable fatty acid-induced cancer cell survival are poorly understood. In this seminar, I will describe our latest findings identifying cytosolic lipid droplets, newly recognized lipid-storage organelles, as the main regulators of unsaturated fatty acid metabolism and resistance to nutrient and oxidative stress. Lipid droplets protect sensitive polyunsaturated fatty acids from oxidation by storing them in the form of inert triglycerides, while simultaneously providing fatty acids for 1) mitochondrial energy production and redox homeostasis and 2) for the synthesis of lipid signaling molecules. Lipid droplets thus emerge as antioxidant and pro-survival organelles that protect cancer cells from stress and are attractive targets for novel therapeutic interventions.

The seminar was held at 16.00 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

23. 02. 2017 Sabina Ott (JSI): Analysis of RNA viruses in invertebrates

Abstract

RNA viruses are the largest group of viruses containing 54 out of 110 families. The majority of viruses causing diseases in humans are RNA viruses, for example: Ebola virus, Zika virus, Coronaviruses (SARS, MERS), Dengue virus, Yellow fever virus, Hepatitis virus, Influenza virus, Rabies virus, Rubella virus, Poliovirus and Measles virus. RNA viruses are classified into three groups, the ss(+)RNA viruses, ss(-)RNA viruses and dsRNA viruses. Their genome sizes are in the range from 2 to 32kb. All RNA viruses encode RNA-dependent RNA polymerase (RdRp). One of the most intriguing feature of viruses is their diversity. Their genome and protein sequences can change beyond recognition over long evolutionary periods. This is due to the high mutation and recombination rates, short generation times, large population sizes and horizontal transfer by vectors. The majority of the studies of the RNA viruses have been done on insects, mammals, fungi and plants. Arthropods have been recognized as the main source of viral diversity. However, numerous invertebrate lineages still remain to be analysed.

To explore the diversity and distribution of RNA viruses in platyhelminths and basal hexapod lineages we have analysed them by homology searching of genome (WGS, nr, Refseq_genomic, Refseq_representative_genomes) and transcriptome (TSA) databases. We have found 12 novel dsRNA viruses that belong to Birnaviridae, Partitiviridae, Reoviridae and Totiviridae 69 ss(+)RNA viruses that belong to Picornavirales, Hepeviridae, Flaviviridae, Narnaviridae, Nodaviridae, Tombusviridae and Negevirus as well as the 48 novel ss(-)RNA viruses that belong to Mononegavirales, Bunyaviridae, Orthomyxoviridae and Quinvirus. Also, 4 ss(+)RNA (Picornavirales, Narnaviridae, Nodaviridae, Tombusviridae) viruses and one ss(-)RNA virus (Mononegavirales) have been found in rotifers. In the genomic databases of platyhelminths and the oldest hexapod lineages we have found two endogenized dsRNA viruses (Partitiviridae), one Negevirus (ss(+)RNA) and 21 endogenized ss(-)RNA viruses. The obtained RNA viral sequences (or complete viral genomes in some cases) have been thoroughly analysed by phylogenetic analysis to confirm their classification into the particular families. The analysis of the genomes of novel complete RNA viruses have shown the differences and similarities with the already known metazoan representatives. This study has shown that platyhelminths and basal hexapod lineages possess quite diverse RNA viromes with numerous novel highly divergent RNA viruses.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

26. 01. 2017 Jernej Šribar (JSI): Ammodytoxin targets R25 in the process of neurotoxicity

Abstract is not available.

The seminar was held at 13.00 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

22. 12. 2016 Valerija Vezočnik (BF, UL): Nanoemulsions of lipid droplets covered by a monolayer of sphingomyelin and cholesterol

Abstract

The research was stimulated by the idea that artificial lipid droplets (LD) named nanoemulsions of LD bearing a polar lipid monolayer surface may serve as a useful lipid membrane model complementary to a vesicle lipid bilayer or lipid monolayer formed at the water/air interface. Nanoemulsions of LD are thermodynamically metastable and kinetically stabilized by a surfactant. Furthermore, they share several physico-chemical characteristics in common with natural LD. These LD have fundamental roles in metabolism, and may also be involved in various diseases. Hence, a well characterized and defined artificial LD system would be convenient because natural systems are usually very complex and less stable. Our study focuses on the characterization of nanoemulsions of LD composed of a trioleoylglycerol (TOG) core coated with a sphingomyelin (SM)/cholesterol (Chol) monolayer. That combination of lipids was chosen since SM and Chol are essential for formation of liquid ordered lipid domains found in both artificial lipid mono- or bilayers and biological membranes. Such domains are implicated in multiple biologically important functions exerted by varied sorts of lipid rafts. LD covered by combined SM and Chol have been poorly characterized in contrast to respective SM/Chol vesicles. Our study aimed at an improved production and characterization of artificial LD composed of TOG, SM and Chol, suitable for the usage in various research areas. A combined modified reverse-phase evaporation/ultrasonication method was developed for preparation of the LD. Structure of nanoemulsions of LD was investigated by using several methods and techniques. LD parameters such as size and shape, and lipid core and monolayer membrane characteristics were determined.

The seminar was held at 14:30 in the lecture room B2 at Biotechnical Faculty (BF) of the University of Ljubljana.

22.-23. 11. 2016 A 2-day Mini-Symposium of the Programme Group (JSI & BF)

On 22 and 23 November we organized a traditional Mini-symposium of our Programme Group. On this occasion members of the group presented their research results and projects. As well, some guests, representatives of the partner groups, presented their projects and joint activities. The event was of an informal and working character to maximally stimulate a critical discussion about the presented results and ideas, aiming to reveal novel collaboration possibilities. The detailed programme of the meeting can be found here.

20.10. 2016 Eva Jarc (JSI): Lipid droplets – key regulators of metabolic and signalling changes in cancer/ Lipidne kapljice - regulatorji metabolnih in signalnih sprememb pri raku

Abstract

Until recently, lipid droplets (LDs) were investigated solely as a lipid storage organelle, however, new evidence suggests that LDs are involved in both metabolic and signalling changes in mammalian cells. Fatty acids (FAs) stored in LDs are released upon triacylglycerol (TAG) lipolysis to provide fuel for mitochondrial β-oxidation and energy production, but also directly affect the regulation of whole cell metabolism through effects on gene expression. Recent studies have shown that lipolysis also provides FAs, such as arachidonic acid (AA), for the synthesis of eicosanoid signalling molecules in human mast cells, suggesting that LDs regulate lipid signalling in inflammatory cells. However, their role as a cellular site for lipid mediator production in cancer or other cells has not been explored yet. Recently, our research group has found a novel mechanism of action of secreted phospholipase A₂(sPLA₂) enzymes in breast cancer cells, which includes an induction of LD formation, changes in cell metabolism and LD-dependent resistance to metabolic stress-induced apoptosis. Interestingly, we also found that sPLA₂ activity on breast cancer cell membranes leads to an incorporation of polyunsaturated fatty acids (PUFAs) into TAGs stored in LDs. We hypothesized that sPLA₂-induced LDs enriched with PUFAs are a source of precursors for eicosanoid production in cancer cells, including the mitogenic and pro-inflammatory prostaglandin E₂ (PGE₂). To show that sPLA₂-induced LDs are a site for PGE₂ production in cancer cells, we have down-regulated LD lipolysis by targeting the main TAG lipase, adipose triglyceride lipase (ATGL) and determined PGE₂ release under various conditions. The second topic, which will be addressed during the seminar, is whether the sPLA₂-induced PUFA enrichment in LDs is important for the pro-survival activity of sPLA₂ in breast cancer cells. Our lipidomic analyses have confirmed that sPLA₂ releases various FAs, including omega-3 and omega-6 PUFAs, from breast cancer cell membranes and induces PUFA enrichment in LDs. To find out which FAs are important for the pro-survival activity of sPLA₂, we performed experiments with exogenously added PUFAs, and compared their actions with that of sPLA₂. To find out if sPLA₂-induced LDs enriched with PUFAs are important for resistance to metabolic stress, we have modulated lipolysis to reduce PUFA release from LDs. Our experiments led us to the surprising finding that ATGL downregulation prevents PUFA lipotoxicity. These and other results will be presented during the seminar to explain why LDs are key regulators of metabolic and signalling changes in cancer.

The seminar was held at 14.30 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

22. 09. 2016 Vesna Hodnik (BF, UL): Identification of specific plant sphingolipids that are receptor molecules for NLP proteins/Identifikacija specifičnih rastlinskih sfingolipidov, ki so receptorske molekule za NLP proteine

Abstract

Main plant pathogens bacteria, fungi, oomycetes, viruses and nematodes cause a number of diseases which affect crop yields and quality of the food production. Pathogens have evolved different molecules to suppress host defense and establish infection despite plant innate immunity. Effectors are all pathogen proteins and small molecules that alter host cell structure and function. A class of conserved secreted effector molecules named NLP proteins can be found in bacteria, fungi and oomycetes. They share sequence similarity with the first described member of this class, Nep1 (Necrosis and ethylene inducing peptide 1), which was isolated from Fusarium oxysporum. They share a conserved NPP1 domain with heptapeptide motif and some conserved cysteine residues. Up until now there are hundreds members known from different species. They trigger immune responses in dicot plants but not in monocots but the mechanism of their action is still not known. It can be either by stimulating host immune responses or by direct cytotoxicity by disrupting the cell membranes. The latter is known as a mode of action for the structurally very similar actinoporins, which are pore-forming toxins from marine invertebrates. Their permeabilizing activity toward membranes is strongly enhanced by the presence of sphingomyelin. Due to structural resemblance between NLP proteins and actinoporins, we predicted the existence of specific sphingolipid receptor molecules for NLPs and molecular mechanism of membrane damage that at least in part resembles that of actinoporins. During the seminar, some recent evidence that NLPs bind to sphingolipids in host membrane will be shown.

The seminar was held at 14:30 in the lecture room B3 at Biotechnical Faculty (BF) of the University of Ljubljana.

16. 06. 2016 Zorica Latinović (JSI): Novel hemostatically active proteins from Vipera ammodytes ammodytes venom: an anticoagulant serine protease homologue and a procoagulant FVIIa-like serine protease / Nova hemostatsko aktivna proteina iz modrasovega strupa: antikoagulantni homolog serinske proteaze in prokoagulantna serinska proteaza, podobna FVIIa:

Abstract

Two proteins with a prominent hemostatic activity, a serine protease homologue, named VaaSPH-1, and a serine protease, SP-10, were purified from the Vipera a. ammodytes venom in a two-step chromatographic procedure using gel filtration and ion-exchange chromatography. As estimated by SDS-PAGE analysis, both proteins are monomers of 34 kDa. They are probably N-glycosylated.

VaaSPH-1 has two mutations in the catalytic triad and is, consequently, enzymatically inactive. In the human plasma, it significantly prolonged the prothrombin blood coagulation time. VaaSPH-1 inhibited the activity of tenase and prothrombinase complex with IC50 of 142 nM and 134 nM, respectively. Using native PAGE analysis we showed that the anticoagulant activity of this molecule is based on its binding to coagulation factors FVII, FIX, FX and prothrombin (FII), and their activated forms. Sequence alignment and 3D structure comparison of these coagulation factors revealed two areas on their surfaces in the vicinity of their active sites where VaaSPH-1 probably binds.

Contrary to VaaSPH-1, enzymatically active SP-10 acted procoagulantly. When added to human plasma, it shortened the activated partial thrombin time and the re-calcification time in a dose-dependent manner. SP-10 specifically activated coagulation FX to FXa, expressing the FVIIa-like activity. It did not bind or activate FIX, FXI and FXII, and did not induce clotting of fibrinogen in the plasma.

Both snake venom proteins are unique and therefore very interesting for further characterization towards the medical usage: VaaSPH-1 as it is a potent non-enzymatic inhibitor of blood coagulation process, and SP-10 as it represents the first known snake venom SP able to specifically activate FX.

The seminar was held at 13.00 in room B220 (2nd floor, Biochemistry building) at the Jožef Stefan Institute (JSI), Jamova cesta 39, Ljubljana.

26. 05. 2016 Vera Župunski (FKKT, UL): ORF1p from LINE1 retrotransposon and its role / Vloga proteina ORF1p iz retrotranspozona LINE1:

Abstract

Transposable elements comprise about half of the human genome and play an important role in genome evolution. Retrotransposon LINE1 (Long INterpersed Element) alone represents 17 % of the human genome and is the only active mobile DNA in humans. Retrotransposition of cis or trans mRNAs has impact on host genome integrity. LINE1 codes for 2 proteins: ORF1p and ORF2p. ORF2p has a endonulease and reverse-transcriptase activity and ORF1p protein binds nucleic acids. The mechanism of retrotransposition is called target primed reverse transcription where mRNA of LINE1 is transported to the cytoplasm, proteins encoded in LINE1 are synthesizes and bound to LINE1 mRNA, the RNP is transported back to the nucleus and LINE1 copy is integrated into the genome.

We are studying nuclear transport of LINE1 and the interactions of ORF1 protein to understand the mechanism and function of LINE1. Specific polyclonal antibodies were raised against N-terminal peptide of ORF1p. Endogenous and overexpressed ORF1p was immunostained in several human cell lines. It was localised mostly in the cytoplasm with anti-tag antibodies. Immunoprecipitation using ORF1p antibodies and anti tag antibodies revealed several protein partners. We have also focused on ORF1p-RNA interactions.

21. 04. 2016 Katja Istenič (BF, UL): Micro- and nanocapsulation of polyphenols into lipid and polysaccharide carriers / Mikro- in nanokapsulacija polifenolov v lipidne in polisaharidne nosilce:

Abstract (in Slovenian language)

Katehini iz zelenega čaja in antocianini iz granatnega jabolka imajo velik potencial za rabo v živilstvu kot nadomestek sintetičnih konzervansov, antioksidantov in barvil. Oviro predstavlja njihova stabilnost pri pogojih, ki jih srečamo med procesiranjem in shranjevanjem živil. Eden od mehanizmov, ki omogočajo zaščito bioaktivnih snovi pred okoljskimi pogoji, je kapsulacija. (±)-katehin in (‒)-epigalokatehin-3-galat (EGCG) smo kapsulirali v liposome, ki smo jih nadalje vključili v mikrodelce iz alginata in hitozana. Tako liposomi kot polisaharidni mikrodelci z vključenimi liposomi so omogočali visoko učinkovitost kapsulacije (84-99 %). S kapsulacijo smo izboljšali stabilnost EGCG pri pH 2,0 in pH 6,0 ter v sadnem nektarju. Za izdelavo submikrometrskih kapsulacijskih sistemov smo uporabili metodo emulzifikacije in na ta način pripravili alginatne delce, velike od 120 do 660 nm, ki so iz senzoričnega vidika bolj primerni za rabo v živilskih izdelkih. Sok granatnega jabolka smo kapsulirali v različne polisaharidne nosilce z metodo liofilizacije. S kapsulacijo smo dosegli olajšano rokovanje s sokom ter zaščito antocianinov pred razgradnjo pri povišani temperature.

24. 03. 2016 Mojca Ogrizović (JSI): Regulation of lipid metabolism and neutral lipid accumulation in yeast Saccharomyces cerevisiae: Its regulation of storage lipids in lipid droplets / Uravnavanje lipidnega metabolizma in kopičenje nevtralnih lipidov pri kvasovki Saccharomyces cerevisiae:

Abstract

Biosynthesis of fatty acids (FA) takes place in the cytosol of the cell. The surplus of FA, which is not needed for the synthesis of cellular components, is stored in lipid droplets as neutral lipids, i.e. triacylglycerols (TAG) and sterol esters (SE). On the other hand, β-oxidation of FA, which in mammals takes place in mitochondria and peroxisomes, is limited only to the latter compartment in yeast Saccharomyces cerevisiae. Although the key enzymes involved in lipid synthesis and degradation are known, a large part of lipid metabolism regulation is still unclear. The research of our group is focused on the regulation of lipid metabolism, in particular on FA β-oxidation regulation through Pex11, a peroxisomal membrane protein, and on the crosstalk of neutral lipid accumulation with central carbon metabolism in yeast S. cerevisiae. We determined Pex11-GFP localization in the background of mutations of all yeast genes (Mattiazzi Ušaj et al., 2015). Pex11-GFP localization was specifically altered by the deletion of cytosol and mitochondrial components of the ER-mitochondria encounter structure (ERMES) complex. Through protein-protein interaction studies we proved that ERMES complex and Pex11 facilitate interaction between mitochondria and peroxisomes. Furthermore, we showed that Pex11 alters expression of specific genes, as well as acetylation of the H4 histone. To understand on a new level neutral lipid accumulation, which is a polygenic trait, extreme quantitative trait loci (X-QTL) mapping method (Ehrenreich et al., 2010) was performed. By comparing a laboratory yeast strain with low, and an industrial strain with high neutral lipid content, we identified three candidate causative genes. Follow-up experiments to elucidate the mechanism by which these genes affect yeast metabolism, including metabolomics analysis and gene editing with CRISPR-Cas9 system will be presented.

Mattiazzi Ušaj M., Brložnik M., Kaferle P., Žitnik M., Wolinski H., Leitner Z., Kohlwein S.D., Zupan B., Petrovič U. (2015): Genome-wide localization study of yeast Pex11 identifies peroxisome–mitochondria interactions through the ERMES complex. JMB 427 (11): 2072-2087

Ehrenreich I.M., Torabi N., Jia Y., Kent J., Martis S., Shapiro J.A., Gresham D., Caudy A.A., Kruglyak L. (2010): Dissection of genetically complex traits with extremely large pools of yeast segregants. Nature 464: 1039-1042

25. 02. 2016 Daša Zupančič (MF, UL): LDL and its role in the regeneration of damaged epithelia / LDL in njegova vloga v regeneraciji poškodovanega epitelija:

Abstract (in Slovenian language)

LDL je lipoproteinski delec namenjen transportu holesterola po telesu. Holesterol je pomembna sestavina celičnih membran, predvsem lipidnih raftov. Celice večinoma privzemajo holesterol od zunaj v obliki LDL delcev, lahko pa ga tudi sintetizirajo. LDL delec je sestavljen iz molekul zaestrenega holesterola, ki jih obdaja fosfolipidni monosloj, v katerem so med molekulami fosfolipidov molekule nezaestrenega holesterola. LDL delci se vnašajo v celice z dobro poznano receptorsko s klatrini posredovano endocitozo. LDL se na zunajcelični strani veže na LDL receptor (LDLR) in velike količine LDLR se skoncentrirajo v pokritih jamicah, tako da je endocitoza zelo učinkovita. LDL v celici potuje po klasični endocitotski poti do razgradnje v lizosomih in prenosa holesterola iz lizosoma v citosol, kjer ga celica lahko uporabi. Holesterol je v celici nujno potreben za preoblikovanje plazmaleme in migracijo celic, proliferacijo celic in oblikovanje medceličnih stikov. Vsi ti procesi pa so ključni za regeneracijo poškodovanega tkiva. S prof. dr. Petrom Veraničem in asist. dr. Natašo Resnik smo raziskali vpliv LDL delcev na regeneracijo poškodovanega epitelija in vitro. Znano je, da so različni tipi celic različno odvisni od zunajceličnega holesterola. Zato smo izbrali dve nerakavi celični liniji z različno odvisnostjo od zunanjega holesterola (MDCK – odvisne in HaCaT – neodvisne) ter tri rakave celične linije (A549, CaCo-2 in RT4). Gojili smo jih v kontrolnem mediju, mediju obogatenem z LDL in mediju brez LDL. Izvedli smo in vitro test reepitelizacije, analizirali razporeditev aktinskih filamentov in tesnih stikov, spremljali proliferacijo in vsebnost ter endocitozo holesterola. Medij obogaten z LDL je spodbudil reepitelizacijo in proliferacijo celic MDCK, medtem ko je bila v mediju brez LDL reepitelizacija statistično značilno nižja. Količina LDL v gojilnem mediju ni imela nobenega vpliva na celice HaCaT. Rakave celične linije so pokazale zelo heterogen odziv na različne medije. Potrebne so nadaljnje študije za ugotavljanje pozitivnih učinkov LDL na regeneracijo epitelijev po poškodbi.

14. 01. 2016 Nuša Trošt (MF, UL): Role of γ-Klotho in triple negative breast cancer / Vloga γ-Klotho pri trojno negativnem podtipu raka dojke:

Abstract (in Slovenian language)

Trojno negativni (TN) rak dojke je ena najbolj agresivnih oblik raka na dojki, ki pogosteje prizadene mlajše bolnice, pogosto metastazira in ima slabo prognozo. Gre za heterogeno skupino rakov, katerim pa je skupno, da ne izražajo receptorjev za estrogen, progesteron in za epidermalni rastni dejavnik tipa 2 (HER2). Zaradi odsotnosti receptorjev se pacientke s tem podtipom raka ne odzivajo na tarčno zdravljenje. Za razvoj tarčnih zdravil je potrebno najti skupne molekularne mehanizme določenih podskupin te heterogene bolezni. Z našo študijo smo ugotovili, da γ-Klotho lahko deluje kot novi označevalec in onkogen za podskupino trojno negativnega raka dojke. S predstavitvijo vam bom podala naša spoznanja kako γ-Klotho, netipični predstavnik koreceptorjev za endokrine fibroblastne rastne faktorje (FGF), vpliva na rast in preživetje TN celic raka dojke ter njegov potencialni mehanizem delovanja.

Past Seminars

17. 12. 2015 Ana Temprano (Hospital Universitari de Tarragona Joan XXIII, Spain): Human lipin family in SGBS adipogenesis

17. 04. 2015 Gregor Gunčar (FKKT, UL): Struktura in uporaba nanoteles

26. 03. 2015 Omar Naneh (KI): Calcium-dependent interaction of perforin with membranes – unravelling old questions using novel techniques

24. 02. 2015 Špela Miklavič (BF, UL): The Pseudomonas aeruginosa RhlR-controlled aegerolysin RahU is a rhamnolipid-binding protein

29. 01. 2015 Miha Mikelj (BF, UL): Interactions of selected MACPF/CDC superfamily proteins with lipid membranes

22. 05. 2014 Matej Skočaj (BF, UL): Labeling of cholesterol-enriched membrane microdomains with ostreolysin A

15. 05. 2014 Minca Ferlin (JSI): Functional and structural characterization of stefins and cystatins from pathogenic bacteria

17. 04. 2014 Jernej Šribar (JSI): Colocalization study of ammodytoxin and its binding proteins in PC12 cell line

27. 03. 2014 Gregor Anderluh (KI): MicroScale Thermophoresis (MST) method

20. 03. 2014 Davor Obradović (BF, UL): Cytolethal distending toxin (CDT) from Aggregatibacter actinomycetemcomitans

13. 03. 2014 Vesna Brglez (JSI): Group X secreted phospholipase A2 is matured intracellularly by furin and proprotein convertase 5

20. 02. 2014 Sabina Berne (BF, UL): Fungal candidate genes implicated in plant pathogenesis identified via transcriptome analyses

13. 02. 2014 Uroš Petrovič (JSI): Yeast Pex11 is involved in the regulation of expression of carbohydrate metabolism genes through chromatin modification by re-routing the acetyl-CoA transport from peroxisomes to mitochondria

06. 02. 2014 Maruša Novak (KI): MACPF and aegerolysins in Aspergillus niger

28. 01. 2014 Angelica Ganga (Laboratory of Biotechnology and Applied Microbiology of the Universidad de Santiago de Chile): Saccharomyces and non-Saccharomyces yeasts in Chilean winemaking

16. 01. 2014 Omar Naneh (KI): Perforin – Pathology, biochemistry and interactions

09. 01. 2014 Nejc Oberčkal (JSI): Ammodytoxin, a neurotoxic snake venom sPLA2, binds specifically to cytochrome c oxidase in mitochondria of target cells

11. 12. 2013 Dušan Kordiš (JSI): Evolutionary genomics and genome biology at IJS

27. 11. 2013 Toni Petan (JSI): Secreted phospholipase A2, lipid metabolism and breast cancer cell survival

06. 06. 2013 Tina Zupančič (KI): The influence of the keratin cytoskeleton on the cell response caused by ionizing radiation and death receptor cytokines

06. 06. 2013 Lidija Kovačič (JSI): Study of complexes between a snake venom or human secreted phospholipase A2 and calmodulin by high-resolution NMR

16. 05. 2013 Špela Miklavič (BF, UL): Regulation of the cellular level of RahU aegerolysin from the bacterium Pseudomonas aeruginosa

13. 02. 2013 Janez Kokošar (JSI): Emergence of novel retroelement-derived domesticated genes: a phylogenomic perspective

25. 01. 2012 Marjetka Podobnik (KI): cAMP mediated cell signaling in mycobacteria

18. 01. 2012 Chiara Gambardella (University of Genoa, Italy): Effect of nanoparticles on fresh water and seawater organisms

11. 01. 2012 Nataša Poklar-Ulrih (BF, UL): Interactions of phenolic compounds with model lipid membranes and proteins

20. 12. 2012 Gregor Anderluh (KI): Update on listeriolysin O research

06. 12. 2012 Adrijana Leonardi (JSI): Discovering innovative drugs and diagnostic tools to control haemostasis by venomics of the Vipera a. ammodytes snake

07. 06. 2012 Matej Skočaj (BF, UL): Interaction of ostreolysin A, a membrane raft binding protein from the oyster mushroom, with natural and artificial lipid membranes

24. 05. 2012 Gorazd Hribar (KI): TNF-α based protein nanoparticles and their use in biomedical applications

17. 05. 2012 Lidija Kovačič (JSI): Probing the allosteric states of the lactose repressor protein

10. 05. 2012 Nada Kraševec (KI): The plant pathogen fungus Cochliobolus lunatus – genome annotation: cytochromes P450, toxins – current status

05. 04. 2012 Sílvia Henriques (Technical University of Lisbon, Lisbon, Portugal): The Saccharomyces cerevisiae Haa1 signaling pathway: structural and functional studies

03. 04. 2012 Miha Mikelj (BF, UL): Interactions of MACPF proteins with lipid membranes

29. 03. 2012 Anja Pucer (JSI): Group X and IIA secreted phospholipases A2 influence proliferation of breast cancer cells

13. 03. 2012 Katja Rebolj (BF, UL): Asymmetrical-flow field-flow fractionation (AF4) and the determination of molar mass and size

08. 03. 2012 Ana Zovko (BF, UL): Biological activities of synthetic analogues of poly-APS

15. 02. 2012 Jernej Šribar (JSI): Novel serum ammodytoxin-binding proteins

24. 01. 2012 Uroš Petrovič (JSI): Identification of quantitative trait loci for high lipid content in Saccharomyces cerevisiae at a single allele resolution

15. 12. 2011 Petra Kaferle (JSI): Towards optimization of yeast colony array phenotyping

31. 05. 2010 Ana Zovko (BF, UL): Biological activities of synthetic analogues of poly-APS

11. 06. 2010 Borut Jerman (JSI): The influence of mammalian sPLA2 on viability of motor-neuron cell line NSC-34

20. 05. 2010 Anja Pucer (JSI): Effects of group X secretory phospholipase A2 overexpression on proliferative and invasive properties of breast cancer cells MDA-MB-231

17. 05. 2010 Damijan Miklavčič (FE, UL): Elektroporacija celičnih membrane

16. 04. 2010 Tamara Sajevic (JSI): Vipera a. ammodytes venom and its effect on the haemostatic system

02. 04. 2010 Mojca Mattiazzi (JSI): Mechanism of inhibition of clathrin-dependent endocytosis by phospholipase A2 in the model organism Saccharomyces cerevisiae

22. 03. 2010 Tilen Praper (BF, UL): Insights into perforin-assisted granzyme delivery

26. 02. 2010 Dušan Kordiš (JSI): Convergent evolution of PLA2s: implications for the classification of the PLA2 “superfamily”

11. 02. 2010 Igor Štagljar (University of Toronto, Canada): Driving biological discoveries using the Membrane Yeast Two-Hybrid (MYTH) technology

15. 01. 2010 Jernej Šribar (JSI): On the effects and mechanism of action of ammodytoxins – study on a model cell line

11. 12. 2009 Adrijana Leonardi (JSI): Structural and biological characterization of Vipera a. ammodytes venom protein components affecting the blood coagulation process

16. 12. 2009 Roman Jerala (KI): Kako Tollu podobni receptorji prepoznavajo patogene

02. 12. 2009 Andrej Bavdek (BF, UL): Aggregation causes listeriolysin inactivation

20. 11. 2009 Janez Kokošar (JSI): Transposable element-derived genes: origin, diversification and gain of novel functions

11. 11. 2009 Urška Žager (University Medical Centre, Ljubljana): Molecular mechanisms of antiphospholipid syndrom

06. 11. 2009 Petra Kaferle (JSI): Towards maximizing the accuracy of agar-based growth fitness measurements

28. 10. 2009 Matej Butala (BF, UL): Activation of bacterial response to DNA damage

26. 05. 2009 Nika Lovšin (JSI): Editors keep genomic editing in check

21. 05. 2009 Ana Zovko (BF, UL): Biological activities of synthetic analogues of poly-APS

21. 04. 2009 Uroš Petrovič (JSI): Imaging-based live cell yeast screen identifies novel factors involved in peroxisome assembly

09. 04. 2009 Tilen Praper (BF, UL): Perforin induces invaginations and formation of secondary vesicels on giant unilamellar vesicles

19. 03. 2009 Špela Konjar (JSI): Role of cathepsins in perforin processing in NK cells and CTLs

26. 03. 2009 Maruša Debeljak (University Medical Centre, Ljubljana): Molekularno genetska diagnostika primarnih imunodeficienc (FHL in CGD)

13. 01. 2009 Vera Župunski (FKKT, UL): Nuclear import of L1 retrotransposon

10. 12. 2008 Biserka Bakrač (BF, UL): Equinatoxin, a eukaryotic pore-forming toxin, as a specific marker for sphingomyelin

25. 11. 2008 Borut Jerman (JSI): Role of endogenous secretory phospholipase A2 in nervous system

12. 11. 2008 Vesna Hodnik (BF, UL): Biacore: Kinetic and affinity analysis

04. 11. 2008 Toni Petan (JSI): Phospholipases A2, arachidonic acid metabolism and cancer

22. 10. 2008 Zoran Arsov (JSI): Supported lipid membranes: Investigation by infrared spectroscopy and possible applications

08. 10. 2008 Osnat Gillor (Ben-Gurion University of the Negev, Israel): Family ties: Bacterial toxins induce their relative’s production

21. 05. 2008 Mojca Podlesnik Beseničar (BF, UL): Perforin and granzyme B on model membranes

16. 04. 2008 Kristina Sepčić (BF, UL): Interactions of a cytolytic protein ostreolysin with cholesterol-enriched membrane domains

19. 03. 2008 Tilen Praper (BF, UL): The properties of human perforin pore

11. 03. 2008 Dušan Kordiš (JSI): Origin of the unique retroelement content in mammalian genomes

31. 01. 2008 Lidija Kovačič (JSI): High-affinity binding of sPLA2s to CaM leads to activation of their enzymatic activity

10. 12. 2007 Jernej Šribar (JSI): Natural ammodytoxin inhibitors

16. 11. 2007 Petra Prijatelj (JSI): Oligomerization of ammodytoxins in solution and in the crystal structure

07. 11. 2007 Andrej Bavdek (BF, UL): Generation of truncated listeriolysin proteins for the study of toxin activity

29. 10. 2007 Mojca Mattiazzi (JSI): High-throughput genetics methods to decipher biological pathways in yeast cells

17. 10. 2007 Matej Butala (BF, UL): Transcriptional regulation of colicin K gene expression

Abbreviations:

BF, Biotechnical Faculty (mostly Dept. Biol.); FE, Faculty of Electrical Engineering; FKKT, Faculty of Chemistry and Chemical Tehnology; JSI, Jožef Stefan Institute (mostly Dept. Mol. Biomed. Sci.); KI, National Institute of Chemistry; MF, Medical Faculty; UL, University of Ljubljana.

B2

Seminars

Forthcoming Programme Group Seminars

Seminars in 2023-2024

Past Seminars

Get in touch

@ Jožef Stefan Institute

Follow our group