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Projects / Programmes source: ARIS

Specificity of interaction of some cytolytic proteins with membrane lipid domains

Research activity

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

Code Science Field
P310  Natural sciences and mathematics  Proteins, enzymology 
P340  Natural sciences and mathematics  Lipids, steroids, membranes 
Keywords
Cytolytic protein; pore-forming protein; lipid membrane; lipid domain; liposome; lipid monolayer; lipid bilayer; unilamellar vesicle; toxin; SPR; EPR; epifluorescence microscopy.
Evaluation (rules)
source: COBISS
Researchers (14)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  15686  PhD Gregor Anderluh  Biochemistry and molecular biology  Researcher  2004 - 2007  969 
2.  27539  PhD Biserka Bakrač Bremec  Biochemistry and molecular biology  Junior researcher  2006 - 2007  29 
3.  21407  PhD Sabina Berne  Biotechnology  Researcher  2004  166 
4.  23477  PhD Mojca Beseničar  Biochemistry and molecular biology  Junior researcher  2004 - 2007  40 
5.  22470  PhD Katarina Črnigoj Kristan  Biochemistry and molecular biology  Junior researcher  2004 - 2005  35 
6.  06994  PhD Peter Maček  Biochemistry and molecular biology  Head  2004 - 2007  523 
7.  12001  PhD Janja Majhenc  Physics  Technical associate  2004 - 2007  73 
8.  17422  Irena Pavešič    Technical associate  2004 - 2007 
9.  27541  PhD Andrej Razpotnik  Natural sciences and mathematics  Junior researcher  2006 - 2007  18 
10.  24291  PhD Katja Rebolj  Neurobiology  Junior researcher  2004 - 2007  57 
11.  12278  PhD Maja Rupnik  Microbiology and immunology  Researcher  2004 - 2007  686 
12.  15328  PhD Kristina Sepčić  Biochemistry and molecular biology  Researcher  2004 - 2007  731 
13.  01119  PhD Marjeta Šentjurc  Biochemistry and molecular biology  Mentor to junior researcher  2004 - 2007  511 
14.  06905  PhD Tom Turk  Biochemistry and molecular biology  Researcher  2004 - 2007  620 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0106  Jožef Stefan Institute  Ljubljana  5051606000  91,005 
2.  0381  University of Ljubljana, Faculty of Medicine  Ljubljana  1627066  48,404 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,847 
Abstract
Binding and pore-formation by cytolytic proteins equinatoxin II (EqtII), a representative of actinoporins, and ostreolysin (Oly) from oyster mushroom, a representative of the aegerolysin protein family, have been shown to be dependant on specific lipid composition of the targeted membrane. There is evidence that EqtII binds and oligomerizes in sphingomyelin containing lipid membranes while preliminary data on the Oly lipid preference reveals specificity for both sphingomyelin and cholesterol. As both lipids are well known to contribute to formation of specific membrane lipid domains, such as lipid rafts, we shall study the proteins binding and pore-forming activity on lipid monolayers, small, large and giant unilamellar lipid vesicles (SUV, LUV, GUV) with respect to occurrence of the specific lipid domains in the model membranes. In comparison, a heteronemertine cytolysin, parborlysin, and bee venom melittin will be used as lipid-nonspecific pore-forming polypeptides. A cytotoxin from the pathogenic bacterium Clostridium difficile, showing lipid.binding and pore-forming activity, will also be analysed for lipid specificity. Lipid domains in SUV and LUV will be detected and characterized by analysis of the line-shape of the electron raramagnetic resonance (EPR) spectra of spin-labelled vesicles, which will give information on order parameter and ratio of diffrent domains in the membrane. Fluorescent lipid probes will be used to visualize specific lipid domains in monolayers and GUV made of binary or ternary lipid mixtures (sphingomyelin, cholesterol, palmitoyl-oleyl-phosphatidylcholine,POPC, or dipalmitoyl-phosphatidyl-choline, DOPC). Alternatively, recombinant EqtII and Oly will be designed to attach fluorescent labels by the thiol chemistry. After binding of the labelled cytolysins, either active or with depressed pore-forming activity, to monolayers and GUV their lateral distribution will be visualized with epifluorescence microscopy and correlated to lipid domain patches reported by domain-specific fluorescent lipid probes (monolayers and GUV). In addition, GUV shapes will be analyzed with respect to the labeled lipid domains and the proteins lateral distribution on the membrane. Based on the fact that either sphingomyelin and cholesterol preferentially distribute into the liquid ordered lipid phase the study is also aimed at developing a fluorescently labeled EqtII and Oly as probes sensing liquid ordered domains in artificial and biological membranes.
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