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Projects / Programmes source: ARIS

Structural explanation of the high increase in enzymatic activity of secreted phospholipases A2 in complex with calmodulin by high resolution NMR.

Research activity

Code Science Field Subfield
1.05.00  Natural sciences and mathematics  Biochemistry and molecular biology   

Code Science Field
P351  Natural sciences and mathematics  Structure chemistry 

Code Science Field
1.04  Natural Sciences  Chemical sciences 
Keywords
secretory phospholipase A2, calmodulin, nonessential activator, neurotoxicity, complex, tridimensional structure, NMR
Evaluation (rules)
source: COBISS
Researchers (1)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  25628  PhD Lidija Kovačič  Biochemistry and molecular biology  Head  2011 - 2013  82 
Organisations (1)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0106  Jožef Stefan Institute  Ljubljana  5051606000  91,836 
Abstract
Secretory phospholipases A2 (sPLA2s) are phospholipid-hydrolysing enzymes, which enzymatic activity is implicated in numerous pathophysiological settings in mammals, for example innate immunity, atherosclerosis, inflammatory diseases, proliferation, apoptosis, removal of damaged cells, cancer, acute respiratory distress syndrome, endotoxic shock, neurotransmitter release, neuritogenesis, embryogenesis, pain, ischemia and Alzheimer’s disease. Tight regulation of their enzymatic activity is therefore of a crucial importance. Ammodytoxin (Atx), a neurotoxic sPLA2, belongs to group IIA sPLA2s and forms a high-affinity complex with cytosolic regulatory protein calmodulin (CaM) which leads to the substantial increase of its enzymatic activity under non-reducing and even more pronouncely under reducing, cytosol-like conditions. The activity enhancement is accompanied, but not caused by conformational stabilization of the enzyme. Based on mapping of the interaction site between Atx and CaM and Atx mutagenesis data we generated energetically most favourable model of Atx–CaM complex. Based on this model we found that CaM induces similar effects also in the case of homologous, physiologically very important, mammalian enzymes, sPLA2s of groups V and X. Following convincing demonstrations that sPLA2 molecules can exist and function also in the cytosol of mammalian cells, we postulated the phospholipase activity-regulating function for CaM. To enable further analysis of the role of CaM in regulation of intra-cytosolic enzyme activity of sPLA2s, a precise tridimensional atomic structure of the sPLA2–CaM complex is needed. In the scope of this project I will use a high-resolution NMR spectroscopy to accomplish this task, as this method can provide us also information about the dynamic changes of the complex on phospholipid membrane resulting in induction of so high increase in sPLA2 enzymatic activity. The detailed tridimensional structure of the complex will enable rational design of either sPLA2 or CaM mutants to be used further in research on pathophysiological relevance of the intracellular action of sPLA2 molecules in mammalian cells. CaM is known to bind proteins in well-established, canonical way. Atx does not possess such CaM-binding motif in its structure and it is speculated that it possesses an original one. By solving the tridimensional structure of the Atx–CaM complex, the Atx`s CaM–binding motif will be unambiguously defined. Then, other proteins with such motifs will be looked for and the role of CaM in their biology tested. Expectedly, the positive results of this project will have high impact on improvement of human health.
Significance for science
SPLA2s are enzymes implicated in numerous pathophysiological processes in mammalian organisms. The extracellular actions of these enzymes have been extensively studied, while the studies of their cellular internalization and action in the cytosol is coming into the focus following our in vitro demonstration of their relative stability in the cytosol-like conditions and demonstration of Atx association with CaM in vivo in the cytosol of a neuronal cell. Our finding that CaM stabilizes and substantially enhances the activity of Atx and some other human sPLA2s provides a possible regulatory mechanism for these enzymes. Knowledge of the CaM interacting surface in the sPLA2-CaM complex offers rational design of mutant on the CaM to be used in research of the pathophysiological relevance of the intracellular action of sPLA2 molecules in mammalian cells. CaM is known to bind proteins in well-established, canonical way through the so called CaM-binding motifs in proteins. By using NMR and modelling we discovered that sPLA2 binds to CaM with new CaM-binding motive, which leads to structural changes in the CaM that seems to be different from any of the canonical CaM-binding protein reported so far. Our result therefore offers the search for new CaM-binding proteins with such CaM-binding motive. Based on the results we predicted and then also verified the functionality of small interfering peptide named MLCKP, which prevented the complex formation between sPLA2 and CaM and, consequently, also the increase and stabilization of enzymatic activity of sPLA2. This peptide presents important tool for further design of new interfering substances that lead to new approaches to interfere/control the CaM-mediated sPLA2 regulatory pathways in mammalian cells. This molecules are highly relevant for our health in the pathophysiological effects mammalian sPLA2s, e.g. presynaptic neurotoxicity, innate immunity, atherosclerosis, inflammatory diseases, proliferation, apoptosis, removal of damaged cells, cancer, acute respiratory distress syndrome, endotoxic shock, neurotransmitter release, neuritogenesis, embryogenesis, pain, ischemia and Alzheimer’s disease.
Significance for the country
The research project had several indirect positive impacts on our society. These were particularly in the transfer of knowledge that I gained in The Netherlands and in the field of education. Through the research activities my scientific and professional experiences grow and my knowledge and experiences were transferred to the students. As a teaching assistant I was involved in teaching activities at undergraduate levels at the Ljubljana University. In the scope of the project one undergraduate and one postgraduate student were directly and practically educated. The results of our research were published in high quality scientific journals and scientific monographs, some results are still in preparation. Together with dissemination of our achievements through invited lectures and on posters on international scientific conferences, I extended the recognition and scientific reputation of Slovenia worldwide. Of the special importance for the Slovenian research sphere and through it for the whole society was also introducing and transferring of the latest research technologies that I used in my experimental work, especially the usage of HADDOCK program and NMR technic.
Most important scientific results Annual report 2011, 2012, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2011, 2012, final report, complete report on dLib.si
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