Amyloid ß-derived switch-peptides as tool to study conformational changes relevant in degenerative diseases

The rapid growing number of patients diagnosed with a neurodegenerative disease and more particularly with Alzheimer's disease (AD) has stimulated intensive research in determining and understanding biological phenomena causing such devastating diseases and hence allowing for the elaboration of adapted therapeutic treatments. These diseases are also commonly called "conformational" diseases because they result from the misfolding of a protein leading to the formation of self-associated β-sheets, which in turn give rise to the formation of oligomers, protofibrils as well as insoluble fibrils characterizing the plaques found in the brain of affected patients. Consequently, the investigation of such proteins, in particular of Amyloid β (Aβ) in the case of AD, is a limited and difficult task to achieve, which often leads to contradictory results. To overcome these difficulties and to be able to study the key steps of conformational transitions and misfolding of such peptides and proteins, our research group has developed a new tool, called switch-peptides, enabling to block (Soff state) and trigger (Son state) peptide folding at will (Figure). The introduction of a switch element S built from Ser, Thr or Cys residues disrupts the regular polypeptide chain by the insertion of an ester and a flexible C-C bond resulting in a conformational disconnection of P1 and P2 (Figure), i.e. in an unordered (random coil), non-folded conformation. Each S element is protected by a protecting group Y (Soff state) that can be cleaved independently by adding a base, an enzyme or by light, depending on the chemical nature of Y. The cleavage of the different protecting groups Y triggers a spontaneous O to N acyl migration, re-establishing the regular amide backbone of the peptide chain, hence enabling the peptide to fold "in statu nascendi" and to adopt a well-defined secondary structure. The present thesis explores the potential of this novel concept for the example of conformational transitions relevant in amyloid β misfolding. In the first part we investigate the chemical stability of the S element in aqueous media, exposing a number of switch-peptides to various experimental conditions. Most notably, the ester bond proved to be stable at acidic as well as physiological conditions for several hours, opening a broad range of biological applications. The second part of the work is dedicated to the study of conformational transitions of switch-peptides derived from Aβ(1-42). By incorporating one or several switch elements disposing orthogonal protecting groups Y, the impact of different fragments of the peptide as nucleation site for the process of β-sheet formation, self-assembly and aggregation has been revealed as monitored by CD, TEM studies and ThT (pathway B, Figure). For the first time, the orthogonal triggering of the two switch elements, i.e. S26 and S37 allowed to delineate the important role of the C-terminal part of Aβ in the early step of misfolding. Subsequently, one of the nucleation sites for aggregation, i.e. segment Aβ(14-24) was excised from the native sequence and transformed to a switch-peptide applying the host-guest technique. Detailed CD studies were applied for investigating conformational transitions of type random-coil (Soff) to β-sheet structure (Son), serving as proof of concept for the use of Aβ-derived nucleation sites as guest sequence in combination with β-sheet promoting host peptides for the screening of potential inhibitors of fibril formation as early molecular event in the context of AD. This has been demonstrated in applying the elaborated host-guest peptides to evaluate the β-sheet breaking potential of pseudo-proline (ψPro)-containing switch-peptides derived from Aβ (pathway C, Figure). Preliminary results indicate that the in situ formation of kink-conformations may exert a β-sheet destabilizing effect, confirming previous observations from the Soto group. Finally, the use of switch-peptides as β-sheet and fibril breaking molecules by in situ α-helix nucleation (pathway A, Figure) has been explored. To this end, the potentially β-sheet forming segment Aβ(14-24) was linked via S element to a helix nucleating peptidomimetic ("N-Cap"). In the Soff state (pH ≤ 4), CD and TEM studies point to the onset of a β-sheet, fibril forming structure. In triggering O,N-acyl migration (pH ≈ 7), a so far unprecedented transition of type β-sheet to α-helix is observed, paralleled by a drastic increase in solubility and a complete disappearance of fibrils. The reversibility of β-sheet and fibril formation by α-helix nucleation in situ represents a most interesting observation and deserves further exploration as potential tool in the study of folding processes. In conclusion, the concept of "switch-peptides" has been successfully applied to biologically relevant molecular events of utmost therapeutic interest.