Vito Foderà

Secondary nucleation and accessible surface in insulin amyloid fibril formation.

At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical study of the fibrillation kinetics suggests that the early stages of the process are affected by stochastic nucleation events.

Thioflavin T Hydroxylation at Basic pH and Its Effect on Amyloid Fibril Detection

The fluorescent dye thioflavin T (ThT) is commonly used for in situ amyloid fibril detection. In this work, we focused on the spectroscopic properties and chemical stability of ThT in aqueous solution as a function of pH, temperature, and dye concentration. A reversible hydroxylation process occurs in alkaline solutions, which was characterized using a combination of UV-vis absorption spectroscopy, proton NMR, and density functional theory (DFT). On the basis of these studies, we propose a chemical structure for the hydroxylated form. Finally, by means of fluorescence spectroscopy, ThT hydroxylation effects on in situ amyloid detection have been investigated, providing new insights on the efficiency of the ThT assay for quantitative fibril evaluation at basic pH.

Self-organization pathways and spatial heterogeneity in insulin amyloid fibril formation.

At high temperature and low pH, the protein hormone insulin is highly prone to form amyloid fibrils, and for this reason it is widely used as a model system to study fibril formation mechanisms. In this work, we focused on insulin aggregation mechanisms occurring in HCl solutions (pH 1.6) at 60 degrees C. By means of in situ Thioflavin T (ThT) staining, the kinetics profiles were characterized as a function of the protein concentration, and two concurrent aggregation pathways were pointed out, being concentration dependent. In correspondence to these pathways, different morphologies of self-assembled protein molecules were detected by atomic force microscopy images also evidencing the presence of secondary nucleation processes as a peculiar mechanism for insulin fibrillation. Moreover, combining ThT fluorescence and light scattering, the early stages of the process were analyzed in the low concentration regime, pointing out a pronounced spatial heterogeneity in the formation of the first stable fibrils in solution and the onset of the secondary nucleation pathways.

Oxidation enhances human serum albumin thermal stability and changes the routes of amyloid fibril formation.

Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimers and Parkinsons diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecular basis of neurodegeneration. In this work, we investigated the effect of hydrogen peroxide oxidation on Human Serum Albumin (HSA) structure, thermal stability and aggregation properties. In the selected conditions, HSA forms fibrillar aggregates, while the oxidized protein undergoes aggregation via new routes involving, in different extents, specific domains of the molecule. Minute variations due to oxidation of single residues affect HSA tertiary structure leading to protein compaction, increased thermal stability, and reduced association propensity.

Factors affecting the formation of insulin amyloid spherulites

Thermally induced amyloid aggregation of bovine insulin can produce a number of distinct aggregate morphologies. In this work amyloid spherulites were analysed using cross polarized optical microscopy and light scattering. A new semi-quantitative methodology to estimate the balance of spherulites and free fibrils is reported and, from this analysis, the effects of pH, temperature, salt, and protein concentration on spherulite formation were quantitatively determined for the first time. The number and size of spherulites measured with polarized light microscopy were related to changes in the colloidal stability of the solution and fibril nucleation times (measured by static light scattering). Importantly, changes in pH between 1.75 and 2 were found to result in a dramatic decrease in the spherulite radii, which were related to differences in the conformational stability of the protein. Moreover, estimates of the final spherulite volume fraction clearly indicate that amyloid spherulite formation is the dominant pathway for insulin aggregation in HCl solutions at low pH and protein concentrations below ~5 mg ml(-1), with the balance shifting towards fibrils as the concentration increases.

Large-scale polymorphism and auto-catalytic effect in insulin fibrillogenesis

Under specific conditions the protein hormone insulin is prone to form amyloid fibrils and is widely used as a model system to study fibril formation mechanisms. In this work we studied thermally induced fibril formation of insulin in acidic solutions. We mainly focused on the effect of the initial protein concentration on the final fibril morphology and the connection between morphology and secondary nucleation mechanisms. Atomic force microscopy (AFM) analysis revealed a significant fibril polymorphism on the macroscopic level dependent on protein concentration, ranging from thin and elongated fibrils to highly complex superstructures. Moreover, amyloid superstructures formed at high insulin concentration resulted in a lower propensity in being stained by Thioflavin T (ThT) and, for these structures, a reduced catalytic effect in seeding experiments was also detected. These results suggest a crucial involvement of the macroscopic properties of fibril surfaces in determining secondary nucleation pathways in insulin fibrillation.

Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth

Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an ‘oligomer stacking model’ for alpha-synuclein fibril elongation.

The route to protein aggregate superstructures: Particulates and amyloid‐like spherulites

Depending on external conditions, native proteins may change their structure and undergo different association routes leading to a large scale polymorphism of the aggregates. This feature has been widely observed but is not fully understood yet. This review focuses on morphologies, physico‐chemical properties and mechanisms of formation of amyloid structures and protein superstructures. In particular, the main focus will be on protein particulates and amyloid‐like spherulites, briefly summarizing possible experimental methods of analysis. Moreover, we will highlight the role of protein conformational changes and dominant forces in driving association together with their connection with the final aggregate structure. Eventually, we will discuss future perspectives in this field and we will comment what is, in our opinion, urgently needed.

Thioflavin T templates amyloid β(1–40) conformation and aggregation pathway

Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimers disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effect of Thioflavin T (ThT), a worldwide used fluorescent dye to monitor amyloid growth, on the Aβ(1-40) conformation, stability and aggregation. By combining experimental information and Molecular Dynamics simulation results, we show that the presence of ThT in solution affects peptide conformation inducing peculiar supramolecular association. In particular ThT interactions with specific Aβ(1-40) residues promote a rigid partially-folded conformation which shifts the balance between different species in solution toward a more aggregation-prone ensemble of peptides, leading to aggregation. Our findings suggest ways for developing strategies to reverse and block aggregation or to stimulate supramolecular assembly and consequently reduce the presence of transient oligomers. This investigation underlines the need of developing label-free techniques for unbiased quantitative studies of Aβ(1-40) aggregation processes.

Tracking the heterogeneous distribution of amyloid spherulites and their population balance with free fibrils.

Abstract.The analysis of amyloidogenic systems reveals the appearance of distinct states of aggregation for amyloid fibrils. For different proteins and under specific experimental conditions, amyloid spherulites are recognized as a significant component occurring in several protein model systems used for in vitro fibrillation studies. In this work we have developed an approach to characterize solutions containing a mixture of amyloid spherulites and individual fibrils. Using bovine insulin as the model system, sedimentation kinetics for the amyloid aggregates were followed using a combination of UV-Vis spectroscopy and cross-polarized optical microscopy. Spherulites were identified as the species undergoing sedimentation. A simple mathematical approach allows the description of the kinetics in terms of decay time/rate distribution. Moreover, based on the sedimentation kinetics, a rough estimate of the balance between amyloid spherulites and individual fibrils can be provided. Fitting the experimental data with the proposed physico-chemical approach shows self-consistent results in reasonable agreement with quantitative imaging analysis previously reported. Our results provide new physical insights into the analysis of amyloidogenic systems, providing a method to characterize the heterogeneous distribution of amyloid spherulites and simultaneously distinguish spherulites and free fibril populations. Importantly, the method can be generally applied to the characterization of polydisperse solutions containing optically traceable spherical particles in the micrometric range.