Projects / Programmes
January 1, 2004
- December 31, 2008
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| P004 |
Natural sciences and mathematics |
Biochemistry, Metabolism |
| P310 |
Natural sciences and mathematics |
Proteins, enzymology |
| P320 |
Natural sciences and mathematics |
Nucleic acids, protein synthesis |
| P340 |
Natural sciences and mathematics |
Lipids, steroids, membranes |
neurotoxic secretory phospholipase A2 (sPLA2), ammodytoxin (Atx), Atx-receptor, membrane pore formers are: equinatoxin, perforin, ostreolysin, parborlysin, alkylpyridinium polymeres, biomembrane, liposome, lipid monolayer, cancer, neuronal system, receptor, synapsa, cytolysis, hemolysis, protein engineering, molecular evolution, retroposon, functional and comparative genomics, yeast.
Researchers (34)
Organisations (3)
Abstract
Several pharmacologically active proteins are already used in biomedicine as a research tool, as a diagnostic or therapeutic resource, and unfortunately also in bio-terrorism. Exact understanding of the mechanism of action of such proteins is a preliminary condition for their useful application and also for defence from them. Secretory phospholipases A2 (sPLA2) are enzymes that have a number of physiological and pathological effects like inflammatory processes, degradation of lipids, cell to cell signalling, defence of organism, they play also an important role in in some cancers. We are particularly interested in ammodytoxin (Atx) a presynaptically neurotoxic sPLA2 of long nosed viper (Vipera ammodytes) venom. Nerve endings exposed to Atx have heavily damaged mitochondria. In plasmalema, omega shaped vesicles are seen that cannot be endocytosed and synaptic vesicles are markedly decreased in number. We are isolating and characterising proteins that bind Atx in neural and other tissues. Our aim is to explain the role of these proteins in neurotoxicity and other (patho)physiological properties. The role of related (endogenic) sPLA2 could be tested for binding to proteins that bind Atx, and may help to explain their still unknown role in the cell. The study of the evolution of the superfamily of phospholipases A2 in eukaryotes will facilitate the understanding of this large group of proteins. The study of adaptive evolution will be continued in several multigene protein families like metalloproteases, inhibitors of serine proteases, perforins and tionins, proteins responsible for defence of different organisms. The research on the evolution of transposable elements (TE) in eukaryots will be continued by searching TE in different genomic sequence data bases that are mostly unexamined ("in silico" approach). Together with evolutionary analyses we will obtain a global insight into the source, evolution and heterogeneity of TE in eukaryots, which is still only fragmentary. Pore forming proteins that have no enzymatic activity to affect biomembranes are among the most wide spread toxins. Cnidarian equinatoxin , human perforin, ostreolysin from mushroom, nemertine's parborlysin and alkylpyridinium polymers from sponge have in common formation of permanent or temporary pores in the lipid part of the membrane. This causes in the cell a series of uncontrolled processes and finally necrotic or apoptotic cell death. Alkylpyridinium polymers from sponge Reniera sarai were found to be inhibitors of acetylcholinesterase and efficient non-detergent pore formers in biological membranes. We shall investigate the possible use of this polymer to protect the underwater surfaces from settling of organisms (so called antifouling).
Significance for science
The research activities that were performed in the scope of our research programme can be broadly classified as toxinology-related research, studies of mammalian sPLA2s, pore forming proteins research, yeast phenomics and genome analysis – these have been complementary and interwoven topics that together build a coherent and powerful research programme. In the field of toxinology our research was focused on the mechanism of action of different components of animal venoms with the aim of developing new molecular tools for physiology and medical research, innovative biological drugs (antithrombics, cytostatics, analgetics, immunotoxins), and for new anti-envenomation approaches (both in terms of detection and prevention/therapy) for medical or anti-bioterrorism applications. The toxins that we have studied have different modes of action and target different cells. We have been focued on the mechanism of action of neurotoxins (especially presynaptically neurotoxic sPLA2s), cytolysins (especially pore-forming proteins and myotoxic sPLA2s), and the venom components affecting haemostasis. The findings on their action on the molecular level are expected to provide an important contribution also in the more basic research of the structure and dynamics of biomembranes (e.g. regulated exocytosis and endocytosis) and protein targeting. They will also contribute to finding new ways to treat thrombo-embolic diseases such as acute heart failure, pulmonary embolism, stroke, and peripheral arterial occlusion. In addition, our research has been aimed at understanding and treating of neurological disorders which result as a consequence of abnormal synaptic vesicle cycling, and regulation of haemostasis. Secreted PLA2s are triggers of signalling cascades (lipid-mediated signalling) and one of the key factors in lipid metabolism in mammals. Understanding these processes is essential for higher-level complexity understanding of diseases and disorders such as Alzheimer’s and Parkinson’s disease, Zellweger syndrome, cardiovascular diseases, atherosclerosis, type 2 diabetes and obesity. Studying the genes encoding long-nosed viper’s venom toxins is also important for explaining the mechanism of enhanced and adaptive evolution of various animal venom protein families. Our research results, among other, is also revealing which regions of the toxin molecules are amenable for development of new functions, without disrupting their 3D structure. Based on these data it will be possible to design proteins with new characteristics, which is crucial for the development of new biologically active molecules. The research topics of our programme group are very relevant and up to date. The results that we published are internationally recognized for their importance.
Significance for the country
Our investigations not only contributed into the world treasury of knowledge, but had also practical values. We developed procedures for production of more efficient and safer antivenoms, novel tumour chemotherapeutics, anticoagulant substances and antithrombotics as well as new tools for intracellular delivery of DNA (alkylpyridinium salts). We developed non-toxic antifouling agents for the protection of the submerged surfaces. Very important aspect of our investigations was the introduction of the latest technologies, especially in the field of functional genomics. Thue we employed diverse novel technologies, such as DNA microarrays, technology of systematic determination of genetic interactions (SGA), latest bioinformatics methods and proteomics. We actively participated in the development of the new research methods. In collaboration with the Slovenian high tech company we developed a manipulator for the automatic replica plating of ordered yeast colonies that enabled us technological advantage and competitiveness at the European level in the analysis of protein toxins and other bioactive substances at the genome level. We participated actively in the development of new bioinformatics tools for the analysis of the results of the genome experiments. We also developed new SPR-based methods for the study of interactions between proteins and lipid membranes. Our research group represents innovative milieu, producing young researchers that are well trained with the latest research techniques of biochemistry, pharmacology, molecular biology and genomics. As such it is very important for Slovenian economy in setting up modern, biotechnology-based companies and laboratories. Therefore our biggest pharmaceutical company Lek-Novartis collaborated intensively with us in the past period. Our research activity was important also for health and defence – in the later we had NATO support for part of our research connected with the war against bioterrorism and the Slovenian Ministry of defence expressed an interest for our development of biosensors for sensitive, quick and mobile detection of chemical and biological toxins. Very important was our participation in the education of young researchers and in the transfer of research-based knowledge to the Slovenian students. Majority of members of our research group is strongly involved into the teaching activities at undergraduate and postgraduate levels, at the Universities of Ljubljana, Maribor and Nova Gorica as well as on the Jožef Stefan International Postgraduate School. Some of our members were holders of Bologna reform-based renovation of faculty programs. A number of young researchers were raised in our group and they moved into the research labs of diverse research institutes and universities, medical clinical laboratories and into the pharmaceutical companies. By numerous publications in high impact scientific journals, invited talks on international conferences, in academic institutions and in industry as well as with the organization of international scientific meetings our research group contributed to the international reinforcement of the reputation of Slovenian science (important part of national culture and originality). Our research group established numerous international collaborations with respectable investigators and institutions around the world. Members of our research group have been performing some of the leading functions in the committees of European and international professional societies (dr. Igor Križaj has been a secretary of a European IST section and the IST board member), which helps in the international recognition of Slovenian science and in strengthening of our national identity. A part of our investigations contributed to the conservation of the richness of our national heritage (of our autochthonic fauna) and to the understanding of the quite big biodiversity, one of the important characteristics of the small Slovenia.
Most important scientific results
Final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Final report,
complete report on dLib.si
