The project is based on the very recent development of a set of ferritin protein mutants endowed with unique properties. In particular a genetically engineered construct has been obtained which corresponds to a truncated Ferritin H-homoplymer (¿Cter-HFt) that lacks 13 aminoacids in the C-terminal region and bears selected mutations in order to abolish cysteine residues in the native positions and introduces a new cysteine in a tolopogically selected position for covalent attachment of bioactive small molecule pendants. ¿Cter-HFt possesses the remarkable capabilities in keeping with ferrooxidase activity while it is not capable of iron sequestering and storage. Thus, in the presence of ferrous iron and oxygen in solution, the protein was demonstrated to catalytically generate a high rate of oxygen radicalic species in vitro. The protein has been fully characterized by CryoEM microscopy in order to demonstrate full and correct 24-mer assembly and tested for cell uptake in ligand tracer measurements. Based on these preliminary observations, the present project aims at demonstrating radical species induced cytotoxicity in a number of selected tumor cell lines (overexpressing transferrin receptor), investigate the oxidative damage on these cells and address at least one animal tumor model for possible tumor cytotoxicity effects.
CURRENT KNOWLEDGE
Mammalian ferritins display a key role in intracellular iron metabolism being capable of oxidizing free ferrous iron and sequestering the resultant ferric iron oxide inside the internal cavity [1]. Thus, ferritin prevents the toxic effect of the oxidative stress induced by free iron within the cell. Lack of ferritin expression is lethal and ferritin mutations that lead to altered iron sequestering activity are at the basis of rare genetic ferritinopathies [2]. In particular, ferritins lacking the C-terminal region have been demonstrated to possess iron ferroxidase activity but are unable to store iron oxide within the internal cavity [3,4].
1: Arosio P, Elia L, Poli M. Ferritin, cellular iron storage and regulation.IUBMB Life. 2017 Mar 27.
2: Vidal R, Delisle MB, Ghetti B. Neurodegeneration caused by proteins with an aberrant carboxyl-terminus. J Neuropathol Exp Neurol. 2004 Aug;63(8):787-800.
3: Luscieti S, Santambrogio P, Langlois d'Estaintot B, Granier T, Cozzi A, PoliM, Gallois B, Finazzi D, Cattaneo A, Levi S, Arosio P. Mutant ferritin L-chains that cause neurodegeneration act in a dominant-negative manner to reduce ferritin iron incorporation. J Biol Chem. 2010 Apr 16;285(16):11948-57.
4: Cozzi A, Corsi B, Levi S, Santambrogio P, Albertini A, Arosio P. Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity. J Biol Chem. 2000 Aug18;275(33):25122-9.
KEY INNOVATION
The fundamental idea behind the project is: why don't we use a ferritin that is intrinsically toxic to the target tumor cells instead of trying loading ferritin with drugs ?
Innovation comes from observations of pathological effects due to some mutations occurring in ferritins that make these molecules unable to sequester iron within the cell thus leading to oxidative damage.