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 lipoxygenase-membrane system from different perspectives and to find answers to the problems still unsolved. In particular, dynamic fluorescence, circular dichroism and infrared spectroscopy will provide information on conformational changes occurring both in the enzyme and membrane structures. Small x ray scattering spectroscopy will be also employed to characterise single lipoxygenase domains changes occurring upon single lipid binding. Light scattering measurements will give information on the size and kinetic of the aggregation process. Finally, fluorescence and atomic force microscopy will be used to visualise preferential interaction of lipoxygenases with raft and non-raft membrane domains. <<<

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 results obtained will provide information for pharmacological drugs design, which could be used to modulate lipoxygenase-induced the oxidative stress.

The study will concern three sets of samples: i) soybean 15-lypoxygenase (15-LOX-1), one of the four lipoxygenases whose crystallographic structure is known and commonly used to mimic the human enzyme; ii) the C-terminal fragment of 15-LOX-1 (mini-LOX), its iron- depleted form (apo-mini-LOX) and reconstituted forms with other metals; iii) the corresponding mammalian enzymes: from rabbit (whose structure is also known), and human lipoxygenase, together with their fragments and mutants.
The research, carried on in collaboration among the three R.U.s will regard: i) the analysis of lipoxygenases conformational changes due to the binding to single lipids; ii) the characterisation of the protein (spontaneous and/or induced) aggregation process; iii) the study of lipoxygenases-induced effects on the structure of lipid bilayers and biological membranes and its partition between raft and non-raft domains.

In order to obtain such information, cellular, biochemical and spectroscopic techniques are required. The R.U.s staff is well trained in the study of the structure-to-function relationship of proteins and in the structural characterisation of biological membranes.
The lipoxygenase-lipids interaction will be studied using six main spectroscopic techniques: fluorescence and circular dichroism (R.U. n.1); ii) Fourier transform infrared spectroscopy (FTIR) and small angle x-ray scattering (SAXS, U.R. n.2); iii) light scattering and atomic force microscopy (AFM, R.U. n.3).
The first four techniques will give detailed information on the various structural aspects (stability, secondary, tertiary and single domain structure) of the different enzymes, on their conformational dynamics (local motility) and on the membranes dynamics; light scattering and AFM measurements will provide quantitative results on the aggregation process and on the lipoxygenases-membranes interaction.
The inter-disciplinary of such approach will allow a characterisation of the specific working mechanism of lipoxygenases and also of the pathologies connected to their disfunctions (due for example to their aggregation) or to their activity itself (oxidative stress damages).
The project will be divided in three phases:
1.a Production of the apo-protein from mini-LOX and of its metal-reconstituted forms.
1.b Cloning of mammals (rabbit and human) lipoxygenase genes.
1.c Characterisation of isothermal compressibility, unfolding/refolding kinetics and aggregation process of soybean 15 lipoxygenase (15-LOX-1).

2.a Production of mammalian recombinant enzymes.
2.b Visualisation of raft and non-raft domains in synthetic membranes.
2.c Structural characterisation of the samples prepared in Phase 1.a, and of their interaction with single lipids as substrate.

3.a Production and study of fragments and mutants of mammalian lypoxygenases.
3.b Interaction between lipoxygenases and membranes: a study of preferential partition of lipoxygenases and their aggregates in different raft and non-raft domains, also as a function of enzymatic activity.

The scientific coordinator has a experience in the coordination of inter-disciplinary projects, both at national and international level, and he recognises the importance of collaboration among the different Research Units, for the success of projects requiring such different but complementary experimental approaches. At this regard, on tops of the regular contacts and exchanges of scientists involved in the project, meetings will be organised every six months for the three responsible scientists of the three R.U., to re-discuss the milestones of the project at the light of the partial results obtained. An important revision and re-assessment of the project will be performed at the end of the first year, checking the progress made in the expression of mammalian enzymes and in the study of soybean lipoxygenase aggregation.
It is also expected a large number of international papers in collaboration among the three R.U.s on international journals, both on the aggregation process and on the effects due to the interaction lipoxygenases-membranes. <<<

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, since they are involved in disfunction and pathologies respectively connected to the cell metabolism of vegetal and animal organisms [2]. They are also a good paradigmatic model of the interaction between cytoplasm proteins and membranes, since their substrate are cell membranes.
Altought intracellular viscosity and molecular crowding can strongly affect the enzymes dinamic properties the development and characterization of preliminar models in vitro is a natural starting point to face the complexity shown by these mechanisms in vivo. The interaction mechanisms between lipoxygenases and lipids and/or model membranes will be studied by the use of tecniques to obtain biochemical (e.g. measuring binding and dissociation constants), structural (e.g. diffusion constants) and dinamic informations (like enzyme-membrane interaction conformational changes).

The enzyme and problems connected to its activity.
Lipoxygenases are citosolyc enzymes widespreaded both in anim al and plant kingdoms [2]. Their preferred substrates are polyunsaturated fatty acids having one or more cis penta dienes. Their biological relevance is due to their double role: they can sinthesize (from single fatty acids) specific bioregulatory idroperoxides and induce membrane structural changes.
Foe instance they are directly involved in plant germination processes [3] and, in mammals, have a key role in leukotrienes and lipotoxins synthesis [4, 5]. In mammals, they are believed to take part also in various deseases like brain aging, HIV infection and cancer [6]. The theoretical background is that their substrates and products can genetically influence cellular metabolism, growth and differentiation [7]. Moreover they have a key role in atherosclerosis development, by interacting with LDL. [8, 9].
It has been demonstrated that regulation of 5-LOX activity is deeply affected by membrane fluidity [10]. In fact lipoxyganase-membrane binding is strongly dependent from its composition, in particular arachidonic acid and its derivatives. Therefore lipoxygenases regulation might be an example of the active role of cellular membranes on metabolism. Indeed, lipidic microdomains, the so-called raft, that are preferred binding sites for membrane-anchored proteins [11], could modulate lipoxygenases activity. On the other hands, these enzymes show aggregation phenomena [12], probably due to exposed idrophobic patches. These exposed idrophobic areas could be involved in the enzyme-membrane interaction, leading to the hypothesis that catalytic activity might be regulated by aggregation. Therefore it is essential to study the aggregation in vitro and its effect on the enzyme interaction with natural and model membranes.
There are various lipoxygenases differing by the arachidonate carbon atom involved in their catalytic cycle. Soybean type 1 lipoxygenases, LOX-1, adding oxygen on carbon C-15, belong to 15-LOXs class. It is one of the few isoenzymes for which a crystallographyc structure is known, and therefore represents a fundamental model for the comprehension of the entire family.

Structure and catalytic mechanism
In spite of the phylogenetic distance and molecular weight differences, vegetable dinatedand animal lipoxygenases could share a similar structure and the same enzymatic catalytic mechanism (2) Actually, all known lipoxygenases are characterized by the same identical catalytic site, made of an iron atom coordinated to four histidine residues and to an isoleucine placed at carboxy-terminal end. The comparison between vegetable LOX-1(13,14) with the correspondent rabbit isoenzyme (whose X-ray structure is known, (15) ) have demonstrated that both proteins are characterized by a N-terminal domain (about 30 kDa) organized almost completely as beta structure and by a C-terminal domain with an high percentage of alpha-helix (about 60 k Da), containing the active site. In accordance with the most accepted model, the entrance of the substrate to the binding site seems to be perpendicular to the entrance of oxygen (2, 16). However, the details of this mechanism are not completely known. The same uncertainty is about the mechanism of substrate acquisition. This latter aspect is among the unresolved questions is important for the comprehension of the general problem regarding the lipid-protein interaction. Among the hypothesis, one is based on the structural similarity between the N-terminal domain of lipoxygenase and the C-terminal domain of some mammal lipases. (15). In this case, the beta structure of C-terminal end is directly involved in the acquisition of lipid substrate. Based on this evidence a similar role has been proposed for the N-terminal end of lipoxygenase.
However, this hypothesis seems not to be true for soybean LOX-1, as recently demonstrated by a recent collaboration study of U.R. n.1 and n.2. The results have demonstrated the the enzyme lacking the N-terminal domain (the 60 kDa fragment named mini-LOX-1) is structural similar to the native protein. Moreover it is able to bind to membranes and shows an enhanced catalytic efficiency (17-19). A deeper insight into the 60 kDa fragment isolated by U.R. n.2 and the comparison with native protein may represent a good initial model for the cmprehension of the interaction mechanism among these proteins and lipids. Moreover, the possibility to obtain the rabbit (20) and human (21) enzymes, could extend such characterization to the mammal lipoxygenases, known to be involved in some pathologies.


Methodologies
The three U.Rs. participating to this project have different expertises in the structural analysis of lipids and proteins The U.R. n.1 and n. 2 have a solid experience on structure and function of lipids and proteins as demonstrated by previous studies regarding lipoxygenases (16) and low density lipoproteins (22). In particular U.R. n. 1 is concerned with dynamic fluorescence spectroscopy and circular dichroism used to study the protein folding, the protein stability as influenced by temperature , ionic strength, pH , etc ) and the structural changes induced by substrate interaction (23-25). By performing measurements under high pressure conditions, the U.R. n.1 is involved in the analysis of elastic properties of proteins such as adiabatic and isothermal compressibility and in the study of consequent volume changes (26,27).

The U.R. n.2 has recently performed a structural analysis of soybean LOX-1 (12) by using small angle x-ray spectroscopy (SAXS). Moreover , by IR spectroscopy (FTIR) the U.R. n. 2 is able to analyse changes in the physical properties of membranes ( foe example in their phase transitions) due to the interaciuon with the enzyme.

The U.R. n.3 ics concerned with static and dynamic light scattering, a fundamental technique for characterization of molecular aggregates. Moreover, the U.R. n.3 has an apparatus for the atomic force spectroscopy (AFM) that due to the ability to analyse millimetric samples is complemental to light scattering spectroscopy (28-309. Moreover, AFM may be used to study the effects produced by lipoxygenase on synthetic and natural membranes.

Finally. some researchers of U.R. n. 1 are specialized in molecular biology and protein purification. These techniques are needed to clone the human lipoxygenase gene and to obtain the recombinant protein and its mutants. <<<