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Folding@home (FAH or F@h) is a distributed computing project aimed to help scientists develop new therapeutics for a variety of diseases by the means of simulating protein dynamics. This includes the process of protein folding and the movements of proteins, and is reliant on simulations run on volunteers' personal computers. Folding@home is currently based at the University of Pennsylvania and led by Greg Bowman, a former student of Vijay Pande. The project utilizes graphics processing units (GPUs), central processing units (CPUs), and ARM processors like those on the Raspberry Pi for distributed computing and scientific research. The project uses statistical simulation methodology that is a paradigm shift from traditional computing methods. As part of the client–server model network architecture, the volunteered machines each receive pieces of a simulation (work units), complete them, and return them to the

Switch-peptides as folding precursors in amyloid fibrillogenesis

Jérémy Bérard, Marie-Stéphanie Camus, Sonia Dos Santos, Richard Alan Mimna, Karine Murat, Lydiane Jeanine Saucède

Intramol. O,N-acyl migration reactions were used as structural switch (S-elements) from a depsipeptide bond contg., unfolded state (Soff) to an all-amide, native states (Son). Chem. or enzymically triggered acyl migrations allowed for the controlled induction of folding events in the process of primary structure evolution, i.e., in statu nascendi (ISN) of the native mol. In the (Soff)-state, the switch-peptide contg. two enzyme (dipeptidyl peptidase IV and pyroglutamate aminopeptidase) cleavable S-elements proved to be highly sol. at physiol. conditions, thus facilitating HPLC purifn. and structural characterization. The conformational decoupling of the individual peptide blocks resulted in a flexible random coil conformation, showing no tendency for self-assocn. or fibril formation after 24 h. In contrast, after selective triggering of the acyl migrations, significant changes in the physicochem. and conformational properties paralleled by the onset of Abeta-like fibrils were obsd.

2006

Le concept switch-peptides

Karine Murat

Conformational transitions have found broad interest due to their impact on protein misfolding and self-assembly as key events leading to the development of neurodegenerative diseases. However, investigation of these dynamic events has been limited so far mainly due to the intrinsic tendency of the involved polypeptides for self-association and aggregation. Consequently, the elucidation of β-sheet and fibril-forming processes related to degenerative diseases remains a difficult task, often giving contradictory results. In order to overcome these difficulties and to investigate early steps of misfolding and subsequent fibril and plaque formation, a new concept termed "Switch-peptides" is presented in this thesis, opening new perspectives for the rational design of drugs preventing Alzheimer's disease. Figure. Elaboration of the "Switch-peptides" concept via S to N acyl migration. As shown in the Figure, the switch-peptides represent stable, self-contained folding precursors, in which folding and self-assembly is blocked by the presence of the Ser-, Thr-, Cys- derived switch elements S dissecting the regular peptide backbone by an (thio)ester and a flexible C-C bond (Soff). Removal of the protecting group Y by enzymatic cleavage or by change of pH, triggers X,N acyl migration restoring the native peptide backbone in situ, and setting off the folding process in the nascent state ("in statu nascendi", ISN) of the parent molecule. These resulting conformational changes i.e. from a random coil to a folded state, can be used to study the onset of biological activity (A) onset (B) and disruption (C) of secondary and tertiary structures. The work described in the present thesis elaborates the structural and chemical foundations of the concept, notably in the induction of structural and conformational properties of peptides. Special attention is focused on the S to N acyl migration in elaborating an in situ ligation methodology. For potential applications in vitro and in vivo, we explore a new thioester solubilizing group to realize efficient native chemical ligations at physiological conditions (Figure). Besides the synthetic access of switch-peptides thioesters via solid-phase techniques, the impact of internal (steric hindrance) and external pH factors on the kinetics of the intramolecular acyl migration is evaluated by analytical HPLC. The major applications of the switch-peptides concept are explored via three main axis : In order to settle the foundations of strategy B, our study was based on the possibility of inducing in situ the formation of secondary and tertiary structures. For this purpose, an amphiphilic model peptide is designed to mimic conformational transitions relevant in degenerative disease, i.e. transitions of type random coil to β sheet. The role of the switch element S in the disruption of secondary structures due to the conformational decoupling of σ on P (Soff), is demonstrated by CD.The induced conformational transition to β sheet conformations is accompanied by a significant decrease in peptide solubility. Moreover, the controlled release of nucleophile within the S element can modulate the rate of structure transition over a broad time scale, as demonstrated by kinetic studies. The use of orthogonal protecting groups Y allows the sequential and individual triggering of multiple elements S, localized in strategic positions in more complex polypeptides. With this objective in mind, the concept is expanded to the induction of ββα supersecondary structures such as the Zinc finger domain (Zif). Actually, the simultaneous introduction of a chemically (pH) and enzymatically (DPPIV) cleavable group at the N- and C-terminus in a sequence derived of Zif1 268 permited the consecutive onset of secondary and tertiary structure by a hierarchical triggering of acyl migrations. The second part of the thesis is devoted to the elaboration of a new generation of β breaking switch-peptides (Figure, strategy C). For the design of such inhibitors, the concept of "separating" recognition (σ inactivated) and functional (σ activated) states appears essential. To construct molecules able to switch between recognition and functional state, we assemble different pseudoproline (ψ Pro)-containing building blocks as induction units σ for β-sheet breaking and a target peptide P. The impact of the ψ Pro-moiety is decoupled from the peptide chain via a flexible thioester bond (Soff) and set off after acyl migration (Son). As a consequence, the insertion and association into existing β-sheet templates should be promoted. Preliminary results by EM studies indicate that a controlled enzyme-induced acyl migration results in a significant improvement of the β-sheet breaking potential. For example, one of the designed switch-peptides slowed down the fibrillogenesis of an amyloid model, representing a significant step in the developement of therapeutically relevant compounds in Alzheimer's disease. The third part focuses on the in situ creation of structure and function of potentially bioactive peptide ligands (Figure, strategy A) via a novel ligation method ("Switch-ligation"). For the example of an angiotensin II-derived analogue, we explore the potential of chemoselective ligations for the onset of biological function under physiologically relevant conditions. Receptor (AT1) binding studies of [Cys5]Ang II in collaboration with PD. Dr. E. Grouzmann, CHUV reveal a fast and efficient ligation without the need of additives (IC50= 25 nM). As an alternative example, the covalent self-assembling of two precursors results in the in situ creation of an efficient peptide deformylase inhibitor. The perspective to extend these methodologies for in vivo applications has been tentatively explored as a new concept in prodrug design. In conclusion, the results of this thesis contribute substantially to the proof of concept of switch-peptides, opening new perspectives in drug design as well as in the study of peptide and protein misfolding relevant in degenerative diseases.

EPFL2006

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

Marie-Stéphanie Camus

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.