Final Activity Report Summary - MEMBRANE-TOK (Transfer of knowledge in biochemistry and molecular biology of cellular membranes in normal cells and in pathological conditions)
The aim of this project was to transfer methodology and principles between laboratories and perform advanced multidisciplinary studies in the field of biomembranes. The research capacities involved lipid biogenesis, molecular genetics and proteomic, synthesis of labelled intermediates and end products, novel model membrane system and methodology for its investigation.
The main scientific subject was to investigate the structure and function of biological membranes, especially the mechanism of their biosynthesis and breakdown. The knowledge in this field is necessary for understanding normal cellular functions and also of basic importance in study of a large group of diseases such as atherosclerosis and diabetes.
Our research, exchange, development of new methodology, workshops, meetings and lectures were concentrated on molecular biological approaches in investigation of gene expression; model membranes for study lipid organisation and function; characterisation and biosynthesis of membrane lipids, especially those of the mevalonate pathway; regulation of membrane biosynthesis by transcriptional factors, and analysis of isolated organelles and subfractionation of organelles by employing immunological, chemical and gradient fractionation methods. The cooperative activities gave several novel results of considerable scientific interest.
The best result was the development of a new principle, the influence of the epoxidated all-trans polyisoprenoids on cholesterol and Coenzyme Q (CoQ) synthesis which is published in the Journal of Biological Chemistry last year and we are presently preparing three publications on this subject. It is also a subject for a common grant application to the Seventh Framework Programme (FP7) of the European Union (submitted in December 2008) and will be the continuation of our future cooperation. Our finding is that certain epoxidated polyisoprenoids upregulate CoQ synthesis and inhibits cholesterol synthesis at the level of oxidosqualene cyclase. These findings are unique and of great medical interest. CoQ is our only endogenously synthesised lipid soluble antioxidant and an important participant in several basic metabolic reactions. Its concentration is decreased during aging and in a number of diseases. On the other hand, increased cholesterol in blood leads to atherosclerosis and heart infarct and this condition today is treated with statins, the most common drug in the Western world. These drugs inhibit the initial part of the mevalonate pathway, and thereby decreasing the synthesis of other important lipids which causes serious side effects. Our epoxidated polyisoprenoids inhibit the terminal part of cholesterol synthesis and therefore, have no deleterious effects. We have performed an extensive study on the biosynthesis, molecular biology, uptake, toxicity and metabolism of these compounds and we have good chances that with cooperation of the pharmaceutical industry develop them to human drugs with valuable new properties.
Several other projects are also completed during the grant period. We have published a new effective carrier system which is able to transfer various non-penetrating substances through the cell membrane. Several linear poly-cis-isoprenoids could be derivatised with heptaprenyltrimethylammonium iodide to form cationic compounds of polyprenols. These compounds proved to be very efficient carriers transferring various cargos into the cell. The efficiency exceeded greatly the capacity of the lipofection agents used today in DNA transfection. We were also successful to synthesise radioactively labelled substrates and intermediates for study the biosynthesis of the mevalonate pathway lipids. The methods described proved to be valuable in study of some genetic disturbances in children with defective synthesis of CoQ causing low values of this lipid in various organs. Methodology for isolation and purification of mitochondria and microsomes from tissue culture cells is also established. The efficiency of the procedures involves that the organelles are in intact form, no soluble components from the inside is leaked out and functions are unaffected.
The main scientific subject was to investigate the structure and function of biological membranes, especially the mechanism of their biosynthesis and breakdown. The knowledge in this field is necessary for understanding normal cellular functions and also of basic importance in study of a large group of diseases such as atherosclerosis and diabetes.
Our research, exchange, development of new methodology, workshops, meetings and lectures were concentrated on molecular biological approaches in investigation of gene expression; model membranes for study lipid organisation and function; characterisation and biosynthesis of membrane lipids, especially those of the mevalonate pathway; regulation of membrane biosynthesis by transcriptional factors, and analysis of isolated organelles and subfractionation of organelles by employing immunological, chemical and gradient fractionation methods. The cooperative activities gave several novel results of considerable scientific interest.
The best result was the development of a new principle, the influence of the epoxidated all-trans polyisoprenoids on cholesterol and Coenzyme Q (CoQ) synthesis which is published in the Journal of Biological Chemistry last year and we are presently preparing three publications on this subject. It is also a subject for a common grant application to the Seventh Framework Programme (FP7) of the European Union (submitted in December 2008) and will be the continuation of our future cooperation. Our finding is that certain epoxidated polyisoprenoids upregulate CoQ synthesis and inhibits cholesterol synthesis at the level of oxidosqualene cyclase. These findings are unique and of great medical interest. CoQ is our only endogenously synthesised lipid soluble antioxidant and an important participant in several basic metabolic reactions. Its concentration is decreased during aging and in a number of diseases. On the other hand, increased cholesterol in blood leads to atherosclerosis and heart infarct and this condition today is treated with statins, the most common drug in the Western world. These drugs inhibit the initial part of the mevalonate pathway, and thereby decreasing the synthesis of other important lipids which causes serious side effects. Our epoxidated polyisoprenoids inhibit the terminal part of cholesterol synthesis and therefore, have no deleterious effects. We have performed an extensive study on the biosynthesis, molecular biology, uptake, toxicity and metabolism of these compounds and we have good chances that with cooperation of the pharmaceutical industry develop them to human drugs with valuable new properties.
Several other projects are also completed during the grant period. We have published a new effective carrier system which is able to transfer various non-penetrating substances through the cell membrane. Several linear poly-cis-isoprenoids could be derivatised with heptaprenyltrimethylammonium iodide to form cationic compounds of polyprenols. These compounds proved to be very efficient carriers transferring various cargos into the cell. The efficiency exceeded greatly the capacity of the lipofection agents used today in DNA transfection. We were also successful to synthesise radioactively labelled substrates and intermediates for study the biosynthesis of the mevalonate pathway lipids. The methods described proved to be valuable in study of some genetic disturbances in children with defective synthesis of CoQ causing low values of this lipid in various organs. Methodology for isolation and purification of mitochondria and microsomes from tissue culture cells is also established. The efficiency of the procedures involves that the organelles are in intact form, no soluble components from the inside is leaked out and functions are unaffected.