Chemical strategies for controlling protein folding and elucidating the molecular mechanisms of amyloid formation and toxicity

It has been more than a century since the first evidence linking the process of amyloid formation to the pathogenesis of Alzheimer's disease. During the last three decades in particular, increasing evidence from various sources (pathology, genetics, cell culture studies, biochemistry, and biophysics) continues to point to a central role for the pathogenesis of several incurable neurodegenerative and systemic diseases. This is in part driven by our improved understanding of the molecular mechanisms of protein misfolding and aggregation and the structural properties of the different aggregates in the amyloid pathway and the emergence of new tools and experimental approaches that permit better characterization of amyloid formation in vivo. Despite these advances, detailed mechanistic understanding of protein aggregation and amyloid formation in vitro and in vivo presents several challenges that remain to be addressed and several fundamental questions about the molecular and structural determinants of amyloid formation and toxicity and the mechanisms of amyloid-induced toxicity remain unanswered. To address this knowledge gap and technical challenges, there is a critical need for developing novel tools and experimental approaches that will not only permit the detection and monitoring of molecular events that underlie this process but also allow for the manipulation of these events in a spatial and temporal fashion both in and out of the cell. This review is primarily dedicated in highlighting recent results that illustrate how advances in chemistry and chemical biology have been and can be used to address some of the questions and technical challenges mentioned above. We believe that combining recent advances in the development of new fluorescent probes, imaging tools that enabled the visualization and tracking of molecular events with advances in organic synthesis, and novel approaches for protein synthesis and engineering provide unique opportunities to gain a molecular-level understanding of the process of amyloid formation. We hope that this review will stimulate further research in this area and catalyze increased collaboration at the interface of chemistry and biology to decipher the mechanisms and roles of protein folding, misfolding, and aggregation in health and disease.

Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins?

Hilal Lashuel

Protein fibrillization is implicated in the pathogenesis of most, if not all, age-associated neurodegenerative diseases, but the mechanism(s) by which it triggers neuronal death is unknown. Reductionist in vitro studies suggest that the amyloid protofibril may be the toxic species and that it may amplify itself by inhibiting proteasome-dependent protein degradation. Although its pathogenic target has not been identified, the properties of the protofibril suggest that neurons could be killed by unregulated membrane permeabilization, possibly by a type of protofibril referred to here as the 'amyloid pore'. The purpose of this review is to summarize the existing supportive circumstantial evidence and to stimulate further studies designed to test the validity of this hypothesis.

Cambridge University Press2006

Studying membrane-protein interplay by modeling realistic conditions

Martina Audagnotto

Realistic models of cellular environments have long captured the imagination of biologists and physical scientists. More than 20 years ago, Goodshellâs inspiring rendering of cellular environments provided the foundation for the idea of capturing the full complexity of biological cells. Since then, increasingly realistic simulations of subcellular models have appeared, showing that we are rapidly moving towards comprehensive, as well as physically and biologically accurate cellular models. The major challenge in developing such models is the necessity to cover a wide range of scales. Indeed, atomistic details of molecular processes are on the length scale of 0.1 nm, while cellular dimensions range from 300 nm (for the smallest bacterial cells) up to 100 ÎŒm (for large eukaryotic cells). The biological range also encompasses the internal dynamics of individual molecules (from ns to ms) as well as entire biological processes (ms to hours). Although an atomistic representation of an entire cell is conceivable, the cost for achieving biologically significant timescales with most advanced computers is very high or nearly impossible currently. Therefore, the combination of in silico approaches with in vivo and in vitro experimental data, allows gaining further insights into the biophysics of the cell. In this thesis, a multi-scale approach was applied to investigate the interplay between proteins and their respective biological environments. In particular, two specific systems were the objects of my Ph.D. project: (i) the amyloid precursor protein (APP) and its interactions at the synaptic plasma membrane, and (ii) the human acyl-protein thioesterase I (hAPT1) and its role in protein depalmitoylation cycle across the Golgi and plasma membranes. Combining atomistic and coarse-grained molecular dynamics (MD) simulations, the stability of the dimeric conformation of APP in a realistic model of the synaptic plasma membrane was investigated. Within this approach, the stable dimeric conformation of the APP as a function of lipid membrane composition was identified, highlighting the important role of lipids unsaturation in protein stability and function. In detail, this indicated not only the preferential dimerization motif of the protein, but also the possible recruiting mode of cholesterol, which has previously been experimentally observed to correlate with Alzheimerâs disease. Furthermore, the role of the synaptic plasma membrane and its ability to form raft microdomains was explored for the interactions of APP with Î³-secretase, which specific catalytic cleavage is responsible for the production of physiological or toxic amyloid Î² peptides, responsible for the Alzheimerâs disease onset. This same multi-scale approach was applied to understand the role of S-acylation in the trafficking and function of hAPT1. In particular, the combination of experimental techniques (i.e., cell biology, biochemistry, microscopy and structural biology) with molecular modeling and simulation disclosed the substrate/enzyme interaction mode, the enzyme/membrane interface, and the role for hAPT1 for a key post-translational modification as S-palmitoylation. Based on our findings, we provided for the first time an atomistic representation of the hAPT1 enzyme mechanism, suggesting that hAPT1 is active in the monomeric form. In conclusion, these studies demonstrate once more the importance of modeling the molecular details of the biological environment at the most accu

EPFL2017

"Switch peptides"

Lydiane Jeanine Saucède

Despite of considerable progress in the research at the interface of Chemistry, Biology and Medicine, neurodegenerative diseases affect more and more seriously our ageing population and remain a real therapeutic challenge for researcher. Nevertheless, advances made in this field allow us to better understand the molecular mechanisms responsible for these disorders and to stress the involvement and the key-role of conformational changes of some proteins. Thought to be at the origin of Alzheimer's disease, amyloid-β peptide undergoes structural modifications leading to β-sheet structures, which further aggregate into toxic fibrils and plaques. However, intrinsic properties of these structures result in problems such as insolubility and self-aggregation limiting their experimental access. Among the main research lines to find an efficient therapeutic agent to fight against Alzheimer's disease, fibrillar inhibition is considered as promising strategy. It requires the use of a molecule, called β-breaker, which would be able to block the misfolding triggered by conformational transitions. Consequently, the present thesis is focusing on the design and chemical synthesis of potential inhibitors of amyloid-β fibrillogenesis. As a specific common feature, the designed compounds integrate in their chemical structure a short sequence ("nucleation site") derived from the central part of Aβ(1-42), allowing to specifically interact with the pathogenic peptide. We first envisaged to design cyclic peptides as potential inhibitors containing proline residues, known for its destabilizing effect upon secondary structures (way A). In collaboration with C. Soto et al., Univ. Galveston, the biological activity of the prototype cyclo(Lys-Leu-Pro-Phe-Phe-Glu) was tested. Most notably, electron microscopy, in collaboration with J. Dubochet and M. Adrian, UNIL, Congo red and thioflavin T staining, and CD studies pointed to the potential of cyclic peptides as Aβ inhibitors. Subsequently, conjugate peptides, assembling the recognition sequence of amyloid-β and organic molecules with potential to block fibrillogenesis were synthesized specifically. (+/-)-Trans-4-cotininecarboxylic acid, 3-indolebutyric acid and a tripeptide β-strand mimic were chosen. The biological assessment of these molecules allowed us to highlight their potential in this therapeutic strategy as well as the advantage given by the intercalation of a proline between the two components of these conjugates. In an effort to introduce in the structure of β-breakers a dynamization element, comparable to the cis/trans isomerization of proline, we designed a new type of β-breaking molecules according to strategy B (Figure). The elaboration of this innovating concept termed "switch-peptides" allows to controll the function of a polypeptide by using intramolecular acyl migration as switch-element for the in situ induction of structure and function. By combining an conformational induction unit σ, a switch element S (cysteine or serine) and an amyloid recognition sequence, we obtained a new generation of dynamic β-breakers. At the Soff state, the σ part was linked to the switch element S via an ester bond, deactivating the structural influence of the conformational induction unit on the target peptide, resulting in the absence of biological activity. By removing the protecting group Y from the amino function of the switch element, intramolecular acyl migration was triggered, restoring the native amide bond, setting off the impact of σ (Son state). In applying pseudo-prolines as β-breaking σ-elements (resulting in a "kink" conformation), the corresponding switch-peptide can adopt a recognition state (Soff) and a β-sheet disrupting state (Son), triggered by controlled acyl migration. We explored the use of cysteine as switch element, with special attention to the chemoselective synthesis, kinetics of acyl migration and potential for β-breaking. Interestingly, the S→N acyl migration proceeded very fast at physiological pH, in comparison to serine derived O→N migrations. Of utmost importance for further extensions of the switch-concept, the design of non peptidic S-elements such as trifunctionalized aromatic compounds would allow us to generate the β-breaking element in situ. As a first step, the intramolecular acyl migration O→N via an intermediate of 5, 6, 7 or 9 membered rings, as well as the reversibility of this reaction, was successfully demonstrated. In addition, enzymatically cleavable protecting groups Y for triggering acyl migrations were established. In conclusion, the present thesis presents some promising concepts in the design of fibril disrupting compounds of considerable therapeutical potential.