α-Synuclein (67-78) (human)

Parkinson's disease-associated α-synuclein is an amyloidogenic protein not only expressed in the cytoplasm of neurons, but also secreted in the extracellular space and internalized into glial cells through a lipid raft-dependent process. We previously showed that α-synuclein interacts with raft glycosphingolipids through a structural motif common to various viral and amyloidogenic proteins. Here we report that α-synuclein also interacts with cholesterol, as assessed by surface pressure measurements of cholesterol-containing monolayers. Using a panel of recombinant fragments and synthetic peptides, we identified two distinct cholesterol-binding domains in α-synuclein. One of these domains, which corresponds to the tilted peptide of α-synuclein (67-78), bound cholesterol with high affinity and was toxic for cultured astrocytes. Molecular modeling suggested that cholesterol binds to this peptide with a tilt angle of 46°. α-synuclein also contains a cholesterol recognition consensus motif, which had a lower affinity for cholesterol and was devoid of toxicity. This motif is encased in the glycosphingolipid-binding domain (34-45) of α-synuclein. In raft-like model membranes containing both cholesterol and glycosphingolipids, the head groups of glycosphingolipids prevented the accessibility of cholesterol to exogenous ligands. Nevertheless, cholesterol appeared to 'signal' its presence by tuning glycosphingolipid conformation, thereby facilitating α-synuclein binding to raft-like membranes. We propose that the association of α-synuclein with lipid rafts involves both the binding of α-synuclein (34-45) to glycosphingolipids, and the interaction of the fusogenic tilted peptide (67-78) with cholesterol. Coincidentally, a similar mechanism is used by viruses (HIV-1, HTLV-I, Ebola) which display a tilted peptide and fuse with host cell membranes through a sphingolipid/cholesterol-dependent process.

Alpha-synuclein is an amyloidogenic protein expressed in brain and involved in Parkinson's disease. It is an intrinsically disordered protein that folds into an alpha-helix rich structure upon binding to membrane lipids. Helical alpha-synuclein can penetrate the membrane and form oligomeric ion channels, thereby eliciting important perturbations of calcium fluxes. The study of alpha-synuclein/lipid interactions had shed some light on the molecular mechanisms controlling the targeting and functional insertion of alpha-synuclein in neural membranes. The protein first interacts with a cell surface glycosphingolipid (ganglioside GM3 in astrocytes or GM1 in neurons). This induces the folding of an alpha-helical domain containing a tilted peptide (67-78) that displays a high affinity for cholesterol. The driving force of the insertion process is the formation of a transient OH-Pi hydrogen bond between the ganglioside and the aromatic ring of the alpha-synuclein residue Tyr-39. The higher polarity of Tyr-39 vs. the lipid bilayer forces the protein to cross the membrane, allowing the tilted peptide to reach cholesterol. The tilted geometry of the cholesterol/alpha-synuclein complex facilitates the formation of an oligomeric channel. Interestingly, this functional cooperation between glycosphingolipids and cholesterol presents a striking analogy with virus fusion mechanisms.

Alpha-synuclein is a 140 residue protein associated with Parkinson's disease. Intraneural inclusions called Lewy bodies and Lewy neurites are mainly composed of alpha-synuclein aggregated into amyloid fibrils. Other amyloidogenic proteins, such as the beta amyloid peptide involved in Alzheimer's disease and the prion protein (PrP) associated with Creuztfeldt-Jakob's disease, are known to possess "tilted peptides". These peptides are short protein fragments that adopt an oblique orientation at a hydrophobic/hydrophilic interface, which enables destabilization of the membranes. In this paper, sequence analysis and molecular modelling predict that the 67-78 fragment of alpha-synuclein is a tilted peptide. Its destabilizing properties were tested experimentally. The alpha-synuclein 67-78 peptide is able to induce lipid mixing and leakage of unilamellar liposomes. The neuronal toxicity, studied using human neuroblastoma cells, demonstrated that the alpha-synuclein 67-78 peptide induces neurotoxicity. A mutant designed by molecular modelling to be amphipathic was shown to be significantly less fusogenic and toxic than the wild type. In conclusion, we have identified a tilted peptide in alpha-synuclein, which could be involved in the toxicity induced during amyloidogenesis of alpha-synuclein.