Structure-properties relationship within highmolecular weight protein oligomeric species associated to Parkinson’s and Huntigton diseases – STRUCTOXIC

Solid state NMR in very high fiels with specific impulses are carried out on alpha-synuclein assemblies fully labeled with heavy carbon, nitrogen and hydrogen isotopes 13C, 15N and 2D. The packing of alpha-synuclein molecules within the different assemblies (intra-molecular interactions) are identified by solid state NMR by mixing fuly labeled 13C alpha-synuclein and 15N alpha-synuclein. The interaction between alpha-synuclein assemblies and specific ligands is also assessed by solid-state NMR. The intra-molecular interaction within alpha-synuclein assemblies is assessed using a cross-linking approach coupled to proteolysis and identification of the cross-linked peptides by mass spectrometry. The same approach is used to map the surface interfaces between alpha-synuclein and the molecular chaperone Hsc70 as we demonstrated that the binding of Hsc70 to alpha-synuclein affects their toxicity and their capacity to propagate from one cell to another.

After sequential assignments of an alpha-synuclein polymorph, a second has been finished by recording and analyzing 3D spectra, including NCACB, NCACO, NCOCA, CANCO and CCC. The two polymorphs show substatial differences in their secondary structures contents. These results have been presented by Julia Gath from the group at ETH Zurich at the ENC conference in Miami, Florida this spring. Beat Meier presented results on alpha-synuclein at the Chianti workshop in Montecatini. Assignments of further polymorphs will be started soon.
We cross-linked Hsc70 to soluble and fibrillar alpha-synuclein assemblies, subjected the cross-links to proteolysis and identified the cross-linked polypeptides. We show that the client binding domain of Hsc70 binds two regions within alpha-synuclein like a tweezer. Our findings define what is necessary and sufficient for engineering Hsc70-derived polypeptide with “minichaperone” properties with a potential as therapeutic agents in Parkinson’s disease through their ability to affect alpha-synuclein assembly and/or toxicity.

Assignments of further polymorphs will be started soon.
Intramolecular interactions identification measurements will be stated soon using the two proposed approaches : SSNMR and cross-linking and mass spectrometry measurements.

Invited Talk:
Protein Structure and Dynamics by Solid-State NMR: Methodological Developments and Application to Fibrils
Beat H. Meier, Anja Böckmann, Matthias Ernst, Ronald Melki, Paul Schanda, Vipin Agarval, Luc Bousset, Carole Gardiennet, Birgit Habenstein, Julia Gath, Matthias Huber, Nina Luckgei, Anders B. Nielsson, Francesco Ravotti, Anne Schütz, Kathrin Szekeley
Chianti Workshop, 17-22.6.2012, Montecatini, Italie

Poster:
The Janus face of a-synuclein
Julia Gath, Birgit Habenstein, Luc Bousset, Ronald Melki, Beat H. Meier, Anja Böckmann
53th ENC Conference, 8-14.4.2012, Miami, Florida

Articles:
The binding of a-Synuclein to human Hsc70 and the yeast Hsp70, Ssa1p: Identification of the molecular chaperone-client protein surface interfaces
Virginie Redeker, Samantha Pemberton, Willy Bienvenut, Luc Bousset, Ronald Melki, Submitted

Parkinson’s and Huntington’s diseases are amongst the most frequent neurodegenerative diseases. These devastating diseases are tightly associated with the assembly into highly ordered high-molecular weight species and propagation between neurons in the central nervous system of these protein aggregates. The two proteins involved in these diseases are alpha-synuclein and huntingtin. We propose to decipher the molecular events leading to alpha-synuclein and huntingtin aggregates formation and characterize the “functional” properties of the different high-molecular weight oligomeric species that form using a combination of cell biological, biochemical and biophysical methods. To link a functional property to a structure, we will characterize alpha-synuclein and huntingtin assemblies at the atomic level using the best-suited method for high-molecular weight oligomeric species, namely solid-state NMR spectroscopy. We will also document the cellular response to alpha-synuclein and huntingtin agregation and propagation alpha-synuclein and huntingtin assemblies.
The structural and functional insights we will gain from our approach will be invaluable for developing therapeutic strategies aimed at modulating the aggregation of proteins involved in two major neurodegenerative diseases that impact the quality of life, lifespan and generate high costs for our developed societies.