INTERACTION STUDIES OF FERRITIN WITH LIPIDS

Maxiferritins are a family of well-characterized iron storage proteins with an essentially ubiquitous distribution in all life forms [1, 2]. They are formed by 24 identical or similar subunits self-assembled into a globular shell containing an iron core consisting of a ferric oxy-hydroxide mineral similar to ferrihydrite [3]. The structure of each subunit is a helix bundle composed of four ?-helices, and a fifth C-terminal short ?-helix. The subunits are assembled into a spherical-shape structure with 4-3-2 symmetry, which forms a hollow complex with an approximately 8 nm diameter cavity capable of storing up to 4500 iron atoms. In vertebrates, native cytosolic ferritins are the product of self-assembly of two types of highly homologous subunits: "light" (L, 20 kDa), and "heavy" (H, 22.8 kDa) subunits, with up to 53% identity. The H subunit is characterized by the presence of a ferroxidase di-iron binding site which is responsible for the enzymatic oxidation of Fe2+ to Fe3+. The L subunit, without a ferroxidase center, still assists Fe2+ oxidation inside the cage, but at a much slower rate, and plays a role in iron nucleation and mineralization [4]. Ferritins display a dynamic iron-storage function, central to cellular iron homeostasis. It has been suggested that, besides its iron-storage function, ferritin plays a role in lipid metabolism [5]. The crystal structure of the HoSF-arachidonate complex (PDB id 4DE6) revealed that the lipid is bound in the pocket at the 2-fold intersubunit contacts. Thus, ferritin may as well protect unsaturated fatty acids from oxidation, limiting their participation in the lipid peroxidation chain reaction, thus reducing their inflammatory effect [5]. Intrigued by the possible role of ferritin as a new fatty acid binding protein we have here investigated the interactions of apo horse spleen ferritin (HoSF) with a pool of lipids, employing a series of 1D 1H-NMR, diffusion (DOSY) and saturation transfer (STD) NMR experiments. Unsaturated fatty acids (arachidonate and oleate) exhibited better binding properties with respect to saturated fatty acids (lauric acid), detergents (sodium dodecyl sulphate) and bile acids (chenodeoxycholic acid). These studies have been accompanied by mineralization assays through UV/visible measurements, aimed at clarifying whether a functional coupling exists between mineralization and ferritin/lipid interactions.