Joint research project

Amyloid aggregation of peptides and proteins on hybrid interfaces (API2)

Project leaders
Fabio Biscarini, Zuzana Gazova
REPUBBLICA SLOVACCA - SAS - Slovak Academy of Sciences
CNR/SAV 2013-2015
Molecular Design
Thematic area
Chemical sciences and materials technology
Status of the project

Research proposal

Our interest is to study the effect of nanoscale confinement on conformational structure, stability and other properties of proteins which are relevant to formation of amyloid structures. Poly-peptides behave differently on surfaces, interfaces or small length scales compared to their bulk properties. Understanding such differences is crucial in systems of medical relevance where proteins are constrained in nanometer size spaces. Examples include dialysis filters, cell crowding, SPECIFY. The results will provide new insights into the effects of soft-matter confinement on protein amyloid fibrillation, a situation often met in natural cell environments.
 The project is focused on the problem of protein aggregation into amyloid structures in relation to environmental factors (temperature, pH, shear flow, ionic strength, surface features, presence of nanoparticles) as it was discovered that the formation of protein amyloid deposits is one of the hallmarks of many very serious diseases. Aggregation imposes also serious restriction to drug dose, storage and distribution as the polypeptides when kept in solution for prolonged periods of time are susceptible to form amyloid aggregates. There is still no effective therapy for these disorders resulting in the increase of medical and social costs and a negative impact in the quality of life of the patient.
 The significant focus in our project will be given to the identification of the low molecular-weight compounds effective in inhibition or destruction of amyloid polymerization. In our study we will investigate several conformationally distinct polypeptides, e.g. random coil peptide (AB peptide – associated with Alzheimer’s disease) and globular proteins (lysozyme - associated with lysozyme amyloidosis; insulin) as prototype systems.
 Most of the studies have been carried out in-vitro under bulk conditions and it was found that formation of the non-native state of proteins can be induced by various factors such as high protein concentration, low protein stability, post-translational modification, presence of toxins, metals, pH, temperature. There are also indications that membrane surfaces or other biosurfaces can cause protein conformational changes which would result into protein fibrillization. Moreover, it was shown that interaction of the proteins with solid surfaces causes extensive alternations in the conformation of the protein backbone, including the loss of the overall protein structure. In human medicine the presence of the artificial surfaces (stents, artificial joints, hemodialyses filters, blood bags, and catheters) can produce conformational changes promoting protein oligomerization through surface forces as well as hydrodynamic or capillary forces. The effect of confinement on aggregation has been not extensively studied to date. In the human body, however, spatial confinement might play an important role, as for instance in the proximity of the extracellular membrane, in synapses, or in multiscale cavities formed by the entanglement of processes and crowding of neural cells.
 Microfabrication involving microfluidics has become a key technology for various biological applications such as drug delivery, 3D cell culture, cell sorting, and biosensors. The main advantages of microfluidics technology include the requirement of minimal amounts of reagents, separations and detections with high resolution/sensitivity, low cost, and short analytical time. This proposal addresses the study of the influence of the confined environment on the aggregation of poly/peptides in vitro. Our aim is to understand how the early stages of amyloid aggregation of proteins can be influenced by the proximity of an interface between the solution and the solid surface. The influence of the interfacial interactions on the aggregation is addressed here by a systematic change of the surface tension of the walls of the microfluidics channels. Microfluidics enables an accurate control the local concentration of biomolecules throughout long incubation times up to hundreds of hours, which is necessary for guaranteeing reproducible conditions.
 Hybrids between bio-molecular and magnetic materials are important for a wide range of biomedical applications such as Magnetic Resonance Imaging (MRI), hyperthermia, cell labeling and sorting, DNA sorting and drug delivery. Nanoparticles possess enormous surface areas due to their small size (tens nm) and it was found out their controversial effect on the protein amyloid aggregation. The current methods for studying amyloid self-assembly are often time-consuming, expensive, and labor intensive, making the analytical process very slow. Thus, a critical need exists for an analytical system that would enable the rapid investigation of amyloid formation with a very small amount of amyloidogenic poly/peptides and reagents. In our project, we would like to find a simple microfluidic biosensor to analyze very small quantities of amyloid aggregates on surface alone and also in presence of various nanoparticles. It was observed that surface composition, size, structure as well as surface charge can markedly change the conformation of protein molecules, and thus influence their propensity to form amyloid structures.
 Comprehensive characterization of amyloid aggregates using spectroscopic and microscopic techniques following the stability and morphology of amyloid aggregation molecular-dynamics modeling of protein aggregation examination of structure, size and morphology of  structures formed after destruction of amyloid aggregates using  experimental techniques (AFM, EM) and mathematical modeling of  segmented objects.
 The vision of the project is to build the knowledge about the mechanisms of formation of protein amyloid aggregates in solution and on the different surfaces. We want to elucidate whether there is a correlation between various conformational states of proteins and their propensity to aggregate as consequence of the properties of the solution and surfaces. By systematically modifying the solution as well as surface parameters will give us precious answers concerning the main factors inducing protein aggregation. Our ansatz is that theextent of protein aggregation can be reduced by carefully controlling both the bulk conditions (e.g., media components, presence of inhibitors of protein aggregation) and surface characteristics (size, surface charge, polarity, tension, structure).

Research goals

Amyloid aggregation of proteins belongs to the serious problems which are still not fully solved. The bilateral cooperation between CNR and SAV allows the integration of the expertise of both scientific teams,aiming to a better understand the processes leading to amyloid aggregation. CNR scientistsare internationally renowned in nanotechnology and nanopatterning of the surfaces., and have extensive experience with microfluidics and AFM on biomolecules. Their role here is mainly in fabrication of geometrically defined environments as well as modulating their surface chemistry and mechanical properties, as well as AFM in liquids. SAV scientists have experience in protein amyloid aggregation, the interaction of amyloid oligomers and fibers with nanoparticles, and data analyses and computer simulations. The collaboration will help to strengthen the relations between the scientists which has already led to significant common scientific results published in international journals, as well as a few European proposals.
The objectives of API-2 are:

understanding/controlling intra/intermolecular interactions involvedin the formation of protein amyloid aggregates in solution;
achieving a comprehensive and quantitative understanding of mechanism of aggregation, adsorption and adhesion activity of relevant proteins in the presence of different surfaces and nanoparticles;
to evaluate the effect of surfaces on protein aggregation process, under different environmental conditions, by advanced imaging techniques combining optical and scanning probe microscopy and its simulation
identification of the compounds able to inhibit or reduce amyloid fibrillization  on the surface and in cell crowding environment
to characterize the conformational propensities of the peptide in solution, we will performed molecular dynamics simulations of a single peptide on different surfaces.

Last update: 27/11/2021