FIBCAT

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

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 Obiettivo del progetto (Objective)

'Protein fibrillation is an important research topic in different scientific fields. For example, increased knowledge on protein fibrillation may clarify different aspects related to pathologies like Alzheimer's and Parkinson's diseases, which are characterized by the build-up of amyloid fibrils in the involved tissues. An important aspect of fibril formation is the ability of fibril itself to catalyze the fibrillation process, known as secondary nucleation, with the fibril surface playing a still poorly understood role in this mechanism. The present proposal aims to investigate aspects of macroscopic fibril polymorphism in relation to the molecular structure of the fibrils and the molecular mechanism of aggregate growth. The focus will be on the surface properties of the fibrils and their role in determining both the evolution of the aggregate growth and the 3D arrangement. Fibril formation will be studied in vitro for two different systems: insulin and alpha synuclein (aSN). The temporal features of the aggregation process will be studied in different experimental conditions using an approach based on amyloid sensitive probes and dynamic light scattering. A morphological investigation on the macroscopic scale of the occurring species will be performed by means of imaging techniques (atomic force and optical microscopy) and the structural details of these species (both intermediate and mature aggregates) will be investigated by small angle X-ray scattering and X-ray fibre diffraction. Surface structural details will be investigated by a novel approach based on a combined use of SAXS and ultrasonic resonator technology, able to provide quantitative information on the compressibility of the solvent around the aggregate. The catalytic propensity of species with different surface properties will be quantified by fluorescence spectroscopy during seeding experiments, and visualized by confocal microscopy.'

Introduzione (Teaser)

Age-related disorders are on the rise with increasing life expectancy. Understanding the pathology and the underlying mechanisms of these conditions is crucial for finding effective therapies.

Descrizione progetto (Article)

The hallmark of many neurodegenerative disorders such as Alzheimer's and Parkinson's disease is the presence of protein aggregates known as amyloids. Amyloid fibrils emerge from the inappropriate folding of various proteins that erroneously interact with each other, causing significant pathologies.

Scientists on the EU-funded FIBCAT project performed a detailed investigation to unravel the mechanism of amyloid formation and their structural features. They used the proteins Concanavalin A, insulin and Equine Lysozime as a model systems, and combined advanced microscopy with small angle X-ray scattering to characterise the aggregates.

Researchers successfully obtained high-resolution information - almost to the level of the atom - of the early structural modifications implicated in protein aggregation. Such detailed knowledge is central for the design of inhibitors to treat neurodegenerative diseases. In addition, they generated data on the physical properties of amyloid structures, focusing on the role of water in inter-protein interactions, the kinetics of aggregation and the morphology of the final aggregates.

Based on these mechanisms, the FIBCAT consortium went on to study the aggregation process for alpha-synuclein, the protein implicated in Parkinson's disease. They investigated the aggregation pathway and the disruptive capacity of various intermediates on biological membranes. They monitored in real time the interaction between protein and membrane to discover that intermediate products were as toxic as final aggregates.

From a therapeutic perspective, researchers used the small molecule thioflavin-T to monitor different conformations and structural states of the protein as well as several fluorescent markers to identify physico-chemical properties of the aggregates.

Interfering with the stability of the aggregated protein and preventing the toxic effects of amyloid fibrils open up novel avenues for treatment of neurodegenerative disorders. Given the detrimental effect of these conditions on the quality of life of patients and their families, the findings of the FIBCAT have great socioeconomic significance.