Real-time Amyloid Aggregation Monitoring with a Photonic Crystal-based Approach

We propose the application of a new label-free optical technique based on photonic nanostructures to real-time monitor the amyloid-beta 1-42 (Ab(1-42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer's Disease (AD). The aggregation of Ab peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Ab peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real-time monitoring of the refractive index variation of the solution, wherein A beta(1-42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages

Real-time Protein Aggregation Monitoring with a Bloch Surface Wavebased Approach

Elsie Barakat, Hans Peter Herzig, Sara Santi

The misfolding and aggregation of amyloid proteins has been associated with incurable diseases such as Alzheimer's or Parkinson's disease. In the specific case of Alzheimer's disease, recent studies have shown that cell toxicity is caused by soluble oligomeric forms of aggregates appearing in the early stages of aggregation, rather than by insoluble fibrils. Research on new strategies of diagnosis is imperative to detect the disease prior to the onset of clinical symptoms. Here, we propose the use of an optical method for protein aggregation dynamic studies using a Bloch surface wave based approach. A one dimension photonic crystal made of a periodic stack of silicon oxide and silicon nitride layers is used to excite a Bloch surface wave, which is sensitive to variation of the refractive index of an aqueous solution. The aim is to detect the early dynamic events of protein aggregation and fibrillogenesis of the amyloid-beta peptide Aβ42, which plays a central role in the onset of the Alzheimer’s disease. The detection principle relies on the refractive index changes caused by the depletion of the Aβ42 monomer concentration during oligomerization and fibrillization. We demonstrate the efficacy of the Bloch surface wave approach by monitoring in real-time the first crucial steps of Aβ42 oligomerization.

Spie-Int Soc Optical Engineering2014

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

Switch peptides

Incorrect folding of proteins, leading to aggregation and amyloid formation, is associated with a group of degenerative diseases including Alzheimer's disease and late onset diabetes. Amyloid forming proteins are believed to be mainly α-helical in their native conformation, but undergo an α-helical to β-sheet conversion paralleled by fibril and plaque formation. As a most challenging experimental hurdle, the transition from helical / rc conformations to β-sheeted structures is followed by dramatic changes in the physicochemical properties, such as self association and insolubility, thus strongly limiting the experimental access of this molecular key process in neurodegenerative diseases. In 2000, M. Mutter and his group proposed a new concept for resolving some of these intrinsic problems of peptide self-assembly. As a basic idea, peptides with a high potential for β-sheet formation, considered as early event in fibrillogenesis, are prepared as 'folding precursors', in which the folding process is blocked by the insertion of a non native bond (see Figure). These modular switch peptides comprise three distinct elements: A conformational induction unit (σ), a switch element (S), and a target peptide (P). A central feature of the concept of switch-peptides, the switch element S dissects the native polyamide backbone of the peptide P by an ester and a flexible C-C bond, thereby preventing the peptide from folding and separating the conformational impact of σ. The triggering of the switch element by removing the protecting group Y reestablishes the native polyamide backbone via a spontaneous O to N acyl migration reaction, whereupon peptide folding can proceed. The present Ph.D. thesis aims to elaborate this concept in putting conformational transitions from helical or random coil to β-sheet structures in focus. This idea is developed using first potentially β-sheet forming model peptides, and is extended in a second step to amyloid-derived sequences. To this end, a series of model peptides exhibiting high β-sheet potential have been synthesized by SPPS synthesis and the critical chain length for β-sheet formation of oligopeptide (Leu-Ser)n was determined by CD studies. The insertion of a pH inducible S-element allowed for the delineation of the in situ transition from rc (Soff) to β-sheet structure (Son). The kinetic evaluation of these conformational transitions as a function of concentration, pH and temperature served as a base for the host-guest peptides according to the switch concept. Host-guest techniques allow to mimic the structural environment of the peptide segments 'excised' from their polypeptide. Typically, peptide segments (guest sequence) regarded as nucleation centers ('hot spots') for β-sheet and fibril formation are incorporated into a host oligopeptide of high β-sheet potential, e.g. (Leu-Ser)n. The incorporation of S-elements at the C- and N-terminal end of the guest segment results in a rc structure (Soff-state), as monitored by CD and TEM studies. By adjusting the pH to physiological conditions, O,N-acyl migration is triggered (Son) and a well defined rc to β-sheet transition is observed by CD, paralleled by the formation of amyloid-like fibrils. For example, after insertion into the β-sheet promoting host peptide (Leu-Ser)n, the amyloid β (14-24) and amylin derived peptide NFGAIL undergo upon pH induced acyl migration a conformational transition from rc to β-sheet , paralleled by the onset of fibril formation. For evaluating the impact of β-breaking compounds upon the kinetics of β-sheet formation, the potential of the developed host-guest switch peptides to serve as a diagnostic tool is elaborated. As the folding is set off in the moment of creating the bioactive molecule ("in statu nascendi", ISN), the present concept allows for the first time to investigate early steps of protein misfolding as relevant in neurodegenerative diseases, opening new perspectives for the rational design of compounds of therapeutic interest. This is demonstrated for the design and synthesis of a reference host-guest system ('molecular kit for screening amyloid β inhibitors'), in which the kinetics of rc to β-sheet transitions is determined under a variety of experimental conditions, including physiological pH. Finally, as a most challenging goal, the disruption or even reversion of β-sheet formation and aggregation is addressed by applying the concept of switch peptides. To accomplish this formidable task, a series of switch-peptides containing various induction systems are investigated. Preliminary results by CD and TEM studies confirm the potential of host-guest switch-peptides for obtaining quantitative data on the impact of β-breaking compounds. A critical evaluation of known β-breaking peptides point to the need for a reliable screening system as developed in the present thesis. The results obtained firmly establish the capability of employing the concept of switch peptides as a tool to study folding/misfolding events during the dynamic process of structure onset and evolution. Keywords: Switch peptides ; host-guest switch peptides ; screening ; inhibitors ; Aβ ; amylin.

EPFL2006

Switch peptides

EPFL2006

Le concept switch-peptides

Karine Murat

EPFL2006