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Direct investigation of the autocatalytic effect in protein fibrillation – from molecular mechanism to macroscopic polymorphism

Final Report Summary - FIBCAT (Direct investigation of the autocatalytic effect in protein fibrillation – from molecular mechanism to macroscopic polymorphism)

This project has addressed a number of open questions in biophysics and structural biology related to protein amyloid self-assembly. Such biological process is at the basis of a number of neurodegenerative pathologies as Alzheimer´s and Parkinson´s diseases. A detailed investigation on the mechanism of formation and the structural features of the final aggregates has been performed for several protein systems, from both a theoretical and an experimental in vitro point of view. We highlight the effect of the flexibility of the protein Concanavalin A on its propensity to aggregate and on its ability to form complex 3D structure. Specifically, we identify the possible formation of several species along the process whose lifetime and stability are strongly dependent on the experimental conditions. The ability to rationalize the occurrence of several species is nowadays crucial for a deeper and effective understanding of the mechanisms involved. We pursued this using a novel approach that combines advanced microscopy analysis and structural characterization of the aggregates using Small Angle X-ray scattering (SAXS). This approach has firstly been applied in the case of Concanavalin A and later to another protein, i.e. equine lysozyme. In the latter case, we further exploited SAXS for an advanced structural characterization that allowed us to isolate the low-resolution structure of an intermediate state during the aggregation pathway. This brought us to gain an almost-atomistic information of the early structural modifications that then lead proteins to aggregate. Such detailed knowledge is for example crucial for the design of potential molecules able to inhibit or reverse the process, being the latter one of the main goal in biomedical sciences for the treatment of neurodegenerative diseases.
We also provided more profound and basic knowledge on the physical properties of a number of amyloid structures. Specifically, investigations on the role of the hydration forces on protein aggregation have been performed, with a specific focus on the hydration state of the final aggregates. The role of water and how it alters the inter-protein interactions during the aggregation process were probed using biophysical methods, structural approaches mainly based on X-ray scattering and theoretical models based on statistical mechanics. Our results show a clear effect of a varied hydration on 1) inter-protein interactions, 2) kinetics (i.e. mechanisms) of aggregation and 3) the final morphologies.
Together with these basic mechanisms behind the aggregation process, we also investigated the aggregation process directly related to both Parkinson´s and Alzheimer´s disease. We systematically studied the aggregation process for alpha-synuclein, a protein involved in the onset of Parkinson´s disease. We studied the formation of intermediate species using a synthetic molecule that helped us to isolate the toxic oligomeric species along the aggregation pathway. The toxicity was investigated via detection of the disruption of model membrane-like systems. The reason for this is that one of the most likely mechanism through which the protein aggregation can affect the biological function of a cell is via membrane disruption. As a consequence using suitable membrane-like systems (in our case liposomes) allows one to probe the toxicity of the isolated species. An advance study on membrane-protein interactions has been also performed for alpha-synuclein in absence of the synthetic molecule. Also in this case we demonstrate the high toxic potency of the intermediate species rather than the one of final aggregate. Moreover, using advanced microscopy analysis, we monitored in real time the mutual interaction between protein and membrane during membrane disruption. This has brought us to visualize the formation of protein-membrane complex during the disruption process that has been also confirmed by SAXS analysis. Regarding the Abeta peptide, a peptide involved in the onset of Alzheimer´s disease, we performed an analysis of the effect of a small molecule, i.e. Thioflavin T, generally used for amyloid detection, on the peptide stability. We verify the possibility to trigger different conformations and structural states of the peptide via the small molecule. In this case we combined experiments and Molecular Dynamics to unravel the basic interactions involved at the level of single aminoacid.

The general result of this project was to apply a multi-perspective approach based on biophysics, biotechnology and pharmaceutical sciences for understanding the molecular basis of amyloid aggregation processes. This is directly connected to the attempt to recover more detail knowledge on the amyloid- related pathologies, a large class of diseases for which no or little treatment is available. Our results provide novel detailed information about the protein-membrane systems, protein energy landscape and on the common generic features at the basis of toxicity. This is of general interest for the understanding of important properties of protein energy landscape and interactions that lead to supra-molecular association and allow, in the long run, guarantying the knowledge for the development of therapeutic strategies for the treatment of neurodegenerative pathologies by a rational screening of small molecules that can prevent amyloid toxic effects in vivo. Neurodegenerative diseases have severe impact on the life quality of both patients and relatives, and represent a significant socio-economical burden. These age-related disorders affect over seven million people in Europe, and this number is expected to further increase as the population ages across Europe. On the light of what stated above, the performed research has fully addressed a much wanted need of the community to effectively face such rapidly increasing problem. Moreover, the realization of the project has also enhanced the know-how within the biotechnological and pharmaceutical industry with a consequent direct and evident economic impact also for the region where the project has been performed. In fact, during the whole duration of the project, a number of scientists within academia and industry have been contacted locally and at European level to be in different extent part of the activities.
Finally, together with the mentioned biomedical relevance, the conducted study on the physic chemical properties, mechanisms of formation and structures is also relevant in material science. In fact amyloid fibrils and superstructures present peculiar properties such as size and structure that suggest their exploitation as new biocompatible materials. Understanding and exploiting protein-based materials is a quickly developing field that will probably drive the future crucial choices within the production sectors of companies developing biodegradable materials. Specifically, one of the main requirements is for these materials having specific and tunable mechanical and optical properties that will allow their use, for example, in tissue engineering. Part of the work points exactly in this direction, demonstrating the possibility of tuning structural and physical properties of the aggregates via subtle changes of the experimental conditions.