Riccardo Pellarin

Prediction of aggregation rate and aggregation-prone segments in polypeptide sequences

The reliable identification of β‐aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimers and Parkinsons diseases, as well as other pathological conditions associated with protein deposition. Here, a model based on physicochemical properties and computational design of β‐aggregating peptide sequences is shown to be able to predict the aggregation rate over a large set of natural polypeptide sequences. Furthermore, the model identifies aggregation‐prone fragments within proteins and predicts the parallel or anti‐parallel β‐sheet organization in fibrils. The model recognizes different β‐aggregating segments in mammalian and nonmammalian prion proteins, providing insights into the species barrier for the transmission of the prion disease.

The role of aromaticity, exposed surface, and dipole moment in determining protein aggregation rates

The mechanisms by which peptides and proteins form ordered aggregates are not well understood. Here we focus on the physicochemical properties of amino acids that favor ordered aggregation and suggest a parameter‐free model that is able to predict the change of aggregation rates over a large set of natural sequences. Furthermore, the results of the parameter‐free model correlate well with the aggregation propensities of a set of peptides designed by computer simulations.

Amyloid aggregation on lipid bilayers and its impact on membrane permeability.

Fibrillar protein aggregates (amyloids) are involved in several common pathologies, e.g., Alzheimers disease and type II diabetes. Accumulating evidence suggests that toxicity in amyloid-related diseases originates from the deposition of protein aggregates on the cell membrane, which results in bilayer disruption and cell leakage. The molecular mechanism of damage to the membrane, however, is still obscure. To shed light on it we have performed coarse-grained molecular dynamics simulations of fibril-forming amphipathic peptides in the presence of lipid vesicles. The simulation results show that highly amyloidogenic peptides fibrillate on the surface of the vesicle, damaging the bilayer and promoting leakage. In contrast, the ordered aggregation of peptides with low amyloidogenicity is hindered by the vesicles. Remarkably, leakage from the vesicle is caused by growing aggregates, but not mature fibrils. The simulation results provide a basis for understanding the range of aggregation behavior that is observed in experiments with fibril-forming (poly)peptides.

Designed Armadillo Repeat Proteins as General Peptide-Binding Scaffolds: Consensus Design and Computational Optimization of the Hydrophobic Core

Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode.

Complete phenotypic recovery of an Alzheimer's disease model by a quinone-tryptophan hybrid aggregation inhibitor.

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimers disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimers disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimers disease.

Amyloid Fibril Polymorphism Is under Kinetic Control

Self-assembly of proteins into amyloid aggregates displays a broad diversity of morphologies, both at the protofibrillar and final fibrillar species. These polymorphic species can coexist at fixed experimental conditions, and their relative abundance can be controlled by changing the solvent composition, or stirring the solution. However, the extent to which external conditions regulate the equilibrium of morphologically distinct species is still unknown. Here we investigate the nucleation of distinct fibril morphologies using computer simulations of a simplified model of an amyloid polypeptide. Counterintuitively, the energetically less favorable fibril morphologies nucleate more frequently than the morphologies of higher stability for models with low aggregation propensity. The free-energy profiles of the aggregation process indicate that the nucleation barrier determines the population fractions of different fibril morphologies, i.e., amyloid polymorphism is under kinetic control.

9,10-Anthraquinone hinders β-aggregation: How does a small molecule interfere with Aβ-peptide amyloid fibrillation?

Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one being Alzheimers disease. In this pathology, the β‐amyloid peptides (Aβ) aggregate into oligomers, protofibrils, and fibrils and eventually into plaques, which constitute the characteristic hallmark of Alzheimers disease. Several low‐molecular‐weight compounds able to impair the Aβ aggregation process have been recently discovered; yet, a detailed description of their interactions with oligomers and fibrils is hitherto missing. Here, molecular dynamics simulations are used to investigate the influence of two relatively similar tricyclic, planar compounds, that is, 9, 10‐anthraquinone (AQ) and anthracene (AC), on the early phase of the aggregation of the Aβ heptapeptide segment H14QKLVFF20, the hydrophobic stretch that promotes the Aβ self‐assembly. The simulations show that AQ interferes with β‐sheet formation more than AC. In particular, AQ intercalates into the β‐sheet because polar interactions between the compound and the peptide backbone destabilize the interstrand hydrogen bonds, thereby favoring disorder. The thioflavin T‐binding assay indicates that AQ, but not AC, sensibly reduces the amount of aggregated Aβ1–40 peptide. Taken together, the in silico and in vitro results provide evidence that structural perturbations by AQ can remarkably affect ordered oligomerization. Moreover, the simulations shed light at the atomic level on the interactions between AQ and Aβ oligomers, providing useful insights for the design of small‐molecule inhibitors of aggregation with therapeutic potential in Alzheimers disease.

Designed armadillo repeat proteins

Designed Armadillo repeat proteins (ArmRPs) are a novel class of binding proteins intended for general modular peptide binding and have very favorable expression and stability properties. Using a combination of sequence and structural consensus analyses, we generated a 42-amino-acid designed Armadillo repeat module with six randomized positions, having a theoretical diversity of 9.9×10(6) per repeat. Structural considerations were used to replace cysteine residues, to define less conserved positions and to decide where to introduce randomized amino acid residues for potential interactions with the target peptide. Based on these concepts, combinatorial libraries of designed ArmRPs were assembled. The most stable version of designed ArmRP in library format was the N5C format, with three randomized library repeat modules flanked by full consensus repeat modules on either side and, in turn, flanked by N- and C-terminal capping repeats. Unselected members of this library were well expressed in the Escherichia coli cytoplasm, monomeric and showed the expected CD spectra and cooperative unfolding. N5C libraries were used in ribosome display selections against the peptide neurotensin. Highly specific peptide binders were enriched after four rounds of selections using ribosome display. Four peptide side chains were shown to contribute most of the interaction energy, and single alanine mutants could be discriminated. Thus, designed ArmRP libraries can become valuable sources for peptide binding molecules because of their favorable biophysical properties and with a potential for application in general modular peptide recognition.

Organism complexity anti-correlates with proteomic β-aggregation propensity

We introduce a novel approach to estimate differences in the β‐aggregation potential of eukaryotic proteomes. The approach is based on a statistical analysis of the β‐aggregation propensity of polypeptide segments, which is calculated by an equation derived from first principles using the physicochemical properties of the natural amino acids. Our analysis reveals a significant decreasing trend of the overall β‐aggregation tendency with increasing organism complexity and longevity. A comparison with randomized proteomes shows that natural proteomes have a higher degree of polarization in both low and high β‐aggregation prone sequences. The former originates from the requirement of intrinsically disordered proteins, whereas the latter originates from the necessity of proteins with a stable folded structure.

Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy

A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42‐residue internal Arm repeats had been designed previously using a sequence‐consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C‐cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature‐ and denaturant‐unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85°C, 14.5° higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.