RICERCA ITALIANA     

Research program

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

University Co-ordinator

Università degli Studi di ROMA "Tor Vergata" - MEDICINA SPERIMENTALE E SCIENZE BIOCHIMICHE - ROMA(RM)

Research Unit Leader

Alessandro FINAZZI AGRO'

Description

The major goals of R.U. n1 will be the determination of protein conformational changes upon ligands binding, the structural modification of lipid bilayers and the partition of lipoxygenases among membranes raft and domains. As described below, this research will be carried on in three different phases. The first one will be dedicated to the samples already available (mini-LOX, apo-mini-LOX and LOX-1) and to the problems connected to their possible aggregated states, in collaboration with R.U. n.3. The second step will concern the characterisation of the metal-substituted enzymes provided by the R.U. in Teramo. Finally, in the last period the effects of these lipoxygenases on synthetic and natural membranes will be studied. A second task of R.U. n.1 will be the expression and purification of the corresponding mammalian enzymes, and in particular the rabbit anf human proteins, which will be possible to express even in mutated forms, to be employed in the study of lipoxygenases-linked pathologies.
This parallel research activity will be also developed into three separate parts.
At the end of each step, a re-examination of the R.U. n1 program will be performed, on the basis of the comparison between the expected and the partial results obtained.

Phase 1
a. folding and stability of mini-LOX and apo-mini-LOX
In this first part of our program we will study in more detail the folding process of mini-LOX, measuring also the stability of apo-mini-LOX, provided by the R.U. n.2. Classical equilibrium-unfolding measurements will be performed using fluorescence and circular dichroism spectroscopy. At this regard, the facilities available are two steady-state fluorometers (K2- ISS, Inc. Champaign, IL, and Fluoromax-3, Jobin Yvon), both equipped with polarizers to measure fluorescence anisotropy, and a spectropolarimeter (model J-700, by Jasco) to measure the protein secondary structure changes in unfolding/refolding experiments. In order to characterise in more detail the folding process of mini-LOX, kinetic studies of the denaturation and refolding process will be performed. In particular, fluorescence and CD measurements will be carried on the above mentioned steady-state fluorometers and spectropolarimeter, recently upgraded with rapid kinetic stopped flow accessories (SFA-20, by Hi-Tech and SFM-20, by Bio-Logic, respectively).
Another problem that will be analysed in this first part of the program in collaboration with R.U. n.3 is the samples aggregation process under different experimental condition, namely protein concentration, ionic strength and temperature. In particular, the aggregation of different mini-LOX samples will be preliminary monitored by R.U. n. 1 with fast scans on a dynamic particle size analyzer (LB-550, by Horiba). Once the aggregation conditions are figured out, samples will be prepared and split in two parts, one of which will be sent to R.U. n.3 for dynamic light scattering measurements, and the second will be analysed by CD and fluorescence spectroscopy, to study the protein conformational changes upon aggregation. Experiments will be carried out in parallel in the two R.U.s (n.1 and n.3) as a function of time, to detect the effects of aging on the protein aggregation state.

b. Cloning of rabbit and human lipoxygenase genes
mRNA from interleukin-13 treated rabbit cells and primary human monocytes will be purified and subjected to RT-PCR in order to obtain the genes coding for rabbit and human lipoxygenases, respectively. The cDNAs will be cloned and sequenced for verification.

Phase 2
a. conformational dynamics of lipoxygenases-ligands interaction
In this second part of the project, the major task of R.U. n. 1 will be the study of the stability and conformational dynamics of the metal-substituted samples prepared by R.U. n.2. Unfolding/refolding studies will be performed as already described in Phase 1. The analysis of the samples dynamics upon ligands binding will be in instead carried on using the dynamic fluorescence methodologies. In particular, we will use the phase-shift and demodulation technique to measure fluorescence lifetimes and rotational correlation lifetimes of the samples in solution. The R.U. n.1 has set up a dynamic fluorometer (model Koala, by ISS) equipped with four laser diodes as excitation sources, both in the UV (i.e. at 280 and 300 nm) and in the visible (i.e. at 370 and 450 nm) region of the spectrum. Samples can be therefore studied both monitoring the intrinsic fluorescence signal and/or the emission of extrinsic probes, such as 1-anilinonaphthalene-8-sulfonic acid (ANS) and 5-dimethylaminonaphtalene-1-sulfonyl chloride (dansyl chloride). Furthermore, enzymatic inhibitors that mimic the binding of the natural lipoxygenase substrates will be used, marked with "ad hoc" fluorescent probes that undergo an energy transfer effect with the tryptophan residues of the protein. In this way fluorescence energy transfer (FRET) can be used to measure intramolecular distances and thus to monitor the conformational changes that characterise the ligand-enzyme binding process. The elastic properties of the lipoxygenases and of the ligand-bound lipoxygenase complex will be characterised measuring the isothermal compressibility of the samples and the associated volume change, up to 3000 and/or 4000 bar, at different temperatures.

b. Expression of rabbit and human lipoxygenases cDNAs
The cDNAs coding for rabbit and human lipoxygenases will be expressed in E. Coli and in a baculovirus/insect cells system, respectively, since purification procedures already exist for the wild type enzymes. Mutant forms will be obtained using commercially available kits.

Phase 3
a. analysis of lipoxygenases interaction and effects on lipid structure and dynamics
The structural and dynamic effects of LOX, mini-LOX and apo-mini-LOX binding to model membranes, either liposomes or unilamellar giant lipid vesicles, will be studied using classical probes such as DPH and Laurdan. In particular, these effects will be monitored through continuous fluorescence lifetime distribution measurements of DPH and through the determination of Laurdan spectral shift, quantitatively assessed with the generalised polarisation function (GP). Enzymatic inhibitors will be used to mimic the simple interaction of enzymes to the membrane in the absence of catalytic activity. The same techniques will be used to verify the effects on membrane structure and dynamics after the oxidative activity of the different enzymes. The results will be compared with those obtained by the R.U. n.2 using the FTIR technique.
On the whole, following the interaction with lipids and the activity of lipoxygenases, these experiments will detect modifications: i) of lipid dynamics in membranes; ii) of the membrane dielectric constant in that this quantity is related to the penetration of water molecule after the production of lipid hydroperoxides; and iii) of lipid domains constituted of different phases.
As a final goal, the verification of lipoxygenases (and possibly lipoxygenases aggregated states) partition coefficients among superficial rafts and/or membranes domains will be taken into examination. This task will be carried on taking also advantage of the atomic force microscopy measurements performed by R.U. n.3.

b. Samples purification
In this last part of the project the recombinant lipoxygenases will be purified together with the obtained mutants. Samples will be therefore ready to be structurally and functionally characterised in collaboration with R.U. n.2 and n.3.

Instruments upgrade
As it appears from the above sections, the fluorescence dynamic instrumentation will play the main role in the development of the R.U. n.1 project. We are therefore planning to upgrade the electronics of the dynamic fluorometer, replacing of the old card with a new and faster data acquisition device. The detection system will be also improved, acquiring a couple of new, high-sensitivity photomultipliers, and the new software package developed by ISS. A new high pressure cell with sapphire windows will be added to the system in order to extend the pressure range up to 4000 bar.
Finally, a fluorescence microscope will be necessary for the study of membrane samples.