Ruolo dei lipid raft nel metabolismo della proteina prionica

The prion protein (PrP) is a GPI-anchored protein primarily concentrated in neuronal cells. Under certain conditions, the innocuous cellular form of this protein, PrPC, can convert into the lethal scrapie isoform, PrPSc, which can aggregate with other PrP molecules and exert its neurotoxic activity. The structure of PrPC consists of two domains: an N-terminal, glycosylated, flexible disordered domain which is capable of binding copper and a C-terminal α-helical domain. In contrast, PrPSc is enriched in β-sheet structures and characterized by its poor solubility in non-denaturing detergents, propensity for aggregation, partial resistance to proteinase K digestion. The conversion of PrPC into PrPSc occurs in particular regions of the cell membrane, enriched with cholesterol and glycosphingolipid, called lipid rafts; these microdomains are thought to play a crucial role both in physiological functions and in the alternative folding of the prion protein. In addition, it’s known that: -PrPC can be cleaved at the 110/111 peptidyl bond to produce a C-terminal fragment, C1, which remains membrane bound and a N-terminal fragment, N1, released in the extracellular space. C1 fragments can’t be converted to the scrapie isoform; - in cell cultures, ADAM10 and ADAM17 were shown to be responsible for this processing and their activation seems PKC-dependent. The aim of our project is to establish if the alteration of cell lipid composition can modify the membrane distribution of the prion protein within rafts or non-raft regions and promote the activity of disintegrins such as ADAM10/17 upon the prion protein. For this reason, granule cells, from the cerebella of 8-day-old rats, were incubated after 8 days in culture with GM1 or GD1a or GT1b for 4 hours at 37°C or with GM1 for 4 hours at 4°C. Detergent resistant fractions, containing lipid rafts, were isolated and proteins in all gradient fractions were separated and analyzed by EF/WB with specific antibodies. After cell treatments with exogenous gangliosides, a good percentage of them was found in lipid rafts; immunoblotting analysis with specific antibodies showed a significant reduction in the amount of proteins, normally localized in lipid rafts, after incubation with GT1b. The incorporation of this ganglioside, characterized by a remarkable steric encumbrance, might be responsible for lipid rafts destabilization and proteins redistribution toward non-raft regions. Another possibility is that GT1b incorporation reduces the number of lipid rafts on the cell membrane. Immunoblotting analysis with three different anti-PrP antibodies showed that this protein is not selectively located in lipid rafts but it is also distributed in several intracellular compartments. Cell treatments with GM1 or GD1a at 37°C for 4 hours were not able to promote PrPC cleavage at the 110/111 peptidyl bond; cell incubation with GM1 seemed able to induce a conformational change of the prion protein toward a “simil-scrapie” isoform, partially resistant to classical denaturation protocols. Further studies are in progress to fully demonstrate that GM1-PrP interaction results in this conformational change.