RICERCA ITALIANA     

Interaction between cytoplasm proteins and membranes: a study of lipoxygenases as a model in vitro and in vivo.

Università degli Studi di Roma "Tor Vergata"

Abstract

Lipoxygenases are key enzymes in the metabolism of both vegetal and animal cells. In mammalian organisms, they are also involved in several serious pathologies but their specific role, at the molecular level is still far to be understood. Some experimental evidences in specific cases, such as AIDS and the Huntington disease, suggest that their interaction with lipid membranes, their natural substrate, may be at the same time the main source of the disfunctions responsible for these diseases. However, it is not known which are the specific consequences on the membrane structure due to the lipoxygenase activity. Furthermore, it is not clear whether a preferential partition of lipoxygenases exists among the different membrane phases and domains. Finally, it has been pointed out that some lipoxygenases display a quite high propensity to aggregation, an effect that might compromise their functionality in vivo.

The aim of this project is to find out answers to these problems. In particular, using a model protein, such as soybean lipoxygenase (15-LOX-1), one of its fragments (mini-LOX) and other iron depleted and metal reconstituted forms, we will study the interaction of lipoxygenases with membranes, also using inhibitors of its enzymatic activity. Cloning of rabbit and human lipoxygenase genes will also allow to extend these studies to mammalian lipoxygenase.

Several complementary techniques will be employed in order to analyse the >>>

Principal Investigator

Alessandro FINAZZI AGRO' Università degli Studi di ROMA "Tor Vergata"

Research Objectives

The target of this project is the structural and functional characterisation of lipoxygenase and of its interaction with lipid substrates.

Under normal circumstances, this family of enzymes plays a key role in both vegetal and animal metabolism. However, in mammals lipoxygenases are directly involved also in serious pathologies, such as cerebral aging, HIV infection and arteriosclerosis. The role of the enzyme in specific diseases (in particular AIDS and Huntington disease) has been identified with its interaction with lipid membranes. However, its specific mechanism at the molecular level has not been yet clarified. For instance, it is not clear whether preferential interactions between the enzyme and particular phases and membrane domains take place. Also it is not known which are the specific changes and damages induced by the enzyme action on the lipid structure of the membrane itself. A second problem concerns the aggregation process, which some lipoxygenase undergo and its correlation to enzymatic disfunctions.

The first part of this project will attempt to find possible explanation to the above-reported questions, studying how the interaction between a model protein (soybean lipoxygenase) and membranes takes place, and also finding how this process is modulated by aggregation. In the second part, the research will be extended also to the corresponding mammalian lipoxygenases available, even with the production of engineered enzymes. The >>>

Timescale

24 months

National and international background

Introduction
The study of complex systems represents one of the major goals of the top scientific research in the field of cell biology. At the molecular level, biological membranes are the most difficult systems to study, for their intrinsic heterogeneity but also because they are dynamic systems, characterised by co-operative chemical and physical properties [1]. In particular, the study of the interaction process between proteins, generally soluble, and membrane lipids represent an important target of the future science, in order to explain important processes involved in cell metabolism, such as signalling, apoptosis and cell trafficking.
With the exception of membrane proteins, that in a few cases display also some hydrophilic domains (as, for instance, in canal-proteins), cytosol enzymes are necessarily provided with an external hydrophilic surface, which confer them the appropriate water solubility. On the other hand, the hydrophobic chains of fatty acids in membranes represent a totally opposite environment, from the chemical and physical point of view. In the mixed system lipids-enzyme the hydrophobic contacts between such different kind of molecules play a key role in the modulation of their reciprocal interaction and thus also in the enzymatic activity. For these reasons the study of these systems requires an interdisciplinary approach.
For our project we have chosen lipoxygenases, enzymes with a strategic relevance in biology and medicine >>>